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CAROTINOIDS  AND 
RELATED    PIGMENTS 

THE  CHROMOLIPOIDS 


BY 


LEROY   S:  PALMER,  Ph.D. 

PEOFESSOR  OF  AGRICULTURAL  BIOCHEMISTRY,   UNIVERSITY  OF  MINNESOTA 


American  Chemical  Society- 
Monograph  Series 


BOOK  DEPARTMENT 
The  CHEMICAL  CATALOG  COMPANY,  Inc. 

19  EAST  24th  STREET,  NEW  YORK,  U.  S.  A. 
1922 


Copyright,  1922,  By 
The  CHEMICAL  CATALOG  COMPANY,  hic. 

All  Rights  Reserved 


Press  of 

J.  J.  Little  &  Ives  Company 

New  York,  U,  S.  A. 


GENERAL  INTRODUCTION 

American  Chemical  Society  Series  of 
Scientific  and  Technologic  Monographs 

By  arrangement  with  the  Interallied  Conference  of  Pure  and  Ap- 
plied Chemistry,  which  met  in  London  and  Brussels  in  July,  1919,  the 
American  Chemical  Society  was  to  undertake  the  production  and 
publication  of  Scientific  and  Technologic  Monographs  on  chemical 
subjects.  At  the  same  time  it  was  agreed  that  the  National  Research 
Council,  in  cooperation  with  the  American  Chemical  Society  and  the 
American  Physical  Society,  should  undertake  the  production  and  pub- 
lication of  Critical  Tables  of  Chemical  and  Physical  Constants.  The 
American  Chemical  Society  and  the  National  Research  Council  mu- 
tually agreed  to  care  for  these  two  fields  of  chemical  development. 
The  American  Chemical  Society  named  as  Trustees,  to  make  the  nec- 
essary arrangements  for  the  publication  of  the  monographs,  Charles 
L.  Parsons,  Secretary  of  the  American  Chemical  Society,  Washington, 
D.  C;  John  E.  Teeple,  Treasurer  of  the  American  Chemical  Society, 
New  York  City;  and  Professor  Gellert  Alleman  of  Swarthmore  Col- 
lege. The  Trustees  have  arranged  for  the  publication  of  the  American 
Chemical  Society  series  of  (a)  Scientific  and  (b)  Technologic  Mono- 
graphs by  the  Chemical  Catalog  Company  of  New  York  City. 

The  Council,  acting  through  the  Committee  on  National  Policy  of 
the  American  Chemical  Society,  appointed  the  editors,  named  at  the 
close  of  this  introduction,  to  have  charge  of  securing  authors,  and  of 
considering  critically  the  manuscripts  prepared.  The  editors  of  each 
series  will  endeavor  to  select  topics  which  are  of  current  interest  and 
authors  who  are  recognized  as  authorities  in  their  respective  fields. 
The  list  of  monographs  thus  far  secured  appears  in  the  publisher's 
own  announcement  elsewhere  in  this  volume. 

The  development  of  knowledge  in  all  branches  of  science,  and  espe- 
cially in  chemistry,  has  been  so  rapid  during  the  last  fifty  years  and 
the  fields  covered  by  this  development  have  been  so  varied  that  it  is 
difiicult  for  any  individual  to  keep  in  touch  with  the  progress  in 

3 


4  GENERAL  INTRODUCTION 

branches  of  science  outside  his  own  specialty.  In  spite  of  the  facilities 
for  the  examination  of  the  literature  given  by  Chemical  Abstracts  and 
such  compendia  as  Beilstein's  Handbuch  der  Organischen  Chemie, 
Richter's  Lexikon,  Ostwald's  Lehrbuch  der  Allgemeinen  Chemie, 
Abegg's  and  Gmelin-Kraut's  Handbuch  der  Anorganischen  Chemie 
and  the  English  and  French  Dictionaries  of  Chemistry,  it  often  takes 
a  great  deal  of  time  to  coordinate  the  knowledge  available  upon  a 
single  topic.  Consequently  when  men  who  have  spent  years  in  the 
study  of  important  subjects  are  willing  to  coordinate  their  knowledge 
and  present  it  in  concise,  readable  form,  they  perform  a  service  of  the 
highest  value  to  their  fellow  chemists. 

It  was  with  a  clear  recognition  of  the  usefulness  of  reviews  of 
this  character  that  a  Committee  of  the  American  Chemical  Society 
recommended  the  publication  of  the  two  series  of  monographs  under 
the  auspices  of  the  Society. 

Two  rather  distinct  purposes  are  to  be  served  by  these  monographs. 
The  first  purpose,  whose  fulfilment  will  probably  render  to  chemists 
in  general  the  most  important  service,  is  to  present  the  knowledge 
available  upon  the  chosen  topic  in  a  readable  form,  intelligible  to 
those  whose  activities  may  be  along  a  wholly  different  line.  Many 
chemists  fail  to  realize  how  closely  their  investigations  may  be  con- 
nected with  other  work  which  on  the  surface  appears  far  afield  from 
their  own.  These  monographs  will  enable  such  men  to  form  closer 
contact  with  the  work  of  chemists  in  other  lines  of  research.  The 
second  purpose  is  to  promote  research  in  the  branch  of  science  covered 
by  the  monograph,  by  furnishing  a  well  digested  survey  of  the  prog- 
ress already  made  in  that  field  and  by  pointing  out  directions  in  which 
investigation  needs  to  be  extended.  To  facilitate  the  attainment  of 
this  purpose,  it  is  intended  to  include  extended  references  to  the  litera- 
ture, which  will  enable  anyone  interested  to  follow  up  the  subject  in 
more  detail.  If  the  literature  is  so  voluminous  that  a  complete  bibli- 
ography is  impracticable,  a  critical  selection  will  be  made  of  those 
papers  which  are  most  important. 

The  publication  of  these  books  marks  a  distinct  departure  in  the 
policy  of  the  American  Chemical  Society  inasmuch  as  it  is  a  serious 
attempt  to  found  an  American  chemical  literature  without  primary 
regard  to  commercial  considerations.  The  success  of  the  venture  will 
depend  in  large  part  upon  the  measure  of  cooperation  which  can  be 
secured  in  the  preparation  of  books  dealing  adequately  with  topics  of 


GENERAL  INTRODUCTION  6 

general  interest;  it  is  earnestly  hoped,  therefore,  that  every  member  of 
the  various  organizations  in  the  chemical  and  allied  industries  will 
recognize  the  importance  of  the  enterprise  and  take  sufficient  interest 
to  justify  it. 


AMERICAN    CHEMICAL    SOCIETY 

BOABD  OF  EDITORS 


Scientific  Series: — 
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Gilbert  N.  Lewis, 
Lafayette  B.  Mendel, 
Arthur  A.  Noyes, 
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Technologic  Series: — 
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C.  G.  Derick, 
William  Hoskins, 

F.  A.  LiDBURY, 

Arthur  D.  Little, 

C.  L.  Reese, 

C.  P.  Townsend. 


American  Chemical  Society 
MONOGRAPH  SERIES 


Other  monographs  in  the  series  of  which  this  book  is  a  part 
now  ready  or  in  process  of  being  printed  or  written. 

Organic  Compounds  of  Mercury. 
By  Frank  C.  Whitmore.    397  pages.    Price  $4.50. 

Industrial  Hydrogen. 
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The  Chemistry  of  Enzym,e  Actions. 
By  K.  George  Falk.     140  pages.    Price  $2.50. 

Ths  V ito/iTivfis . 
By  H.  C.  Sherman  and  S.  L.  Smith.    273  pages.    Price  $4.00. 

The  Chemical  Effects  of  Alpha  Particles  and  Electrons. 
By  Samuel  C.  Lind.     180  pages.    Price  $3.00. 

Zirconium  and  Its  Compounds. 
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The  Properties  of  Electrically  Conducting  Systems. 
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The  Analysis  of  Rubber. 
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The  Origin  of  Spectra. 
By  Paul  D.  Foote  and  F.  L.  Mohler.    Price  $4.50. 

Thyroxin.    By  E.  C.  Kendall. 

The  Properties  of  Silica  and  the  Silicates.    By  Robert  B.  Sosman. 

Coal  Carbonization.    By  Horace  C.  Porter. 

The  Corrosion  of  Alloys.    By  C.  G.  Fink. 

Piezo-Chemistry .    By  L.  H.  Adams. 

Cyanamide.    By  Joseph  M.  Braham. 

Liquid  Ammonia  as  a  Solvent.    By  E.  C.  Franklin. 

Wood  Distillation.    By  L.  F.  Hawley. 

Solubility.    By  Joel  H.  Hildebrand. 

Glue  and  Gelatin.    By  Jerome  Alexander. 

Organic   Arsenical  Compounds.    By   George   W.  Raiziss.     Jos- 
eph L.  Gavron. 

Valence,  and  the  Structure  of  Atoms  and  Molecules.    By  Gil- 
bert N.  Lewis. 

Shale  Oil.    By  Ralph  H.  McKee. 

Aluminothermic  Reduction  of  Metals.     By  B.  D.  Saklatwalla. 

The  Chemistry  of  Leather  Manufacture.    By  John  A.  Wilson. 

Absorptive  Carbon.    By  N.  K.  Chaney. 

Refining  Petroleum.    By  George  A.  Burrell,  et  al. 

Extraction  of  Gasoline  from  Natural  Gas.  By  George  A.  Burrell. 

The  Animal  as  a  Converter.    By  H.  P.  Armsby  and  C.  Robert 
Moulton. 

Chemistry  of  Cellulose.    By  Harold  Hibbert. 

The  CHEMICAL  CATALOG  COMPANY,  Inc. 

19  EAST  24th  STEEET,  NEW  YOEK,  U.  S.  A. 


PREFACE 

Color  in  nature  may  properly  be  divided  into  two  groups,  namely, 
color  due  to  structure,  which  is  caused  by  light  reflections  from  col- 
loidal particles  of  air  or  water,  and  color  due  to  pigments,  which  is 
caused  by  substances  having  remarkable  powers  of  absorption  of  light 
rays  of  certain  wave  length  and  reflection  of  others.  The  reflected 
rays,  of  course,  give  the  pigment  its  color. 

The  present  monograph  treats  of  pigmented  substances  having  a 
yellow,  yellow-orange,  orange,  red-orange  and  red  color.  So  far  as  the 
author  is  aware  no  authentic  instances  of  structural  colors  of  these 
hues  have  been  reported.  In  fact,  the  wave  length  of  light  in  these 
regions  of  the  spectrum  is  probably  too  great  for  such  a  phenomenon 
to  occur  for  colloidal  emulsions  having  the  refractive  index  of  air  or 
water.  The  particular  pigments  to  be  considered  are  widely  dis- 
tributed in  every  stage  of  living  matter,  and  are  perhaps  more  fre- 
quently encountered  than  any  other  class  of  natural  pigments.  They 
have  attracted  the  attention  of  the  biologists  for  at  least  100  years. 
Among  the  earliest  inquiries  were  those  of  Caventau  (1817)  and 
Goebel  (1823).  The  former  was  interested  in  the  yellow  pigment  of 
the  daffodil,  the  latter  in  the  pigments  of  the  crab  and  in  the  feet  of 
doves  and  geese. 

Active  investigation  of  these  pigments  in  plants  and  animals  has 
been  confined  to  the  past  fifty  years.  It  has  only  been  within  the  past 
fifteen  years,  however,  that  the  chemical  composition  of  any  of  these 
pigments  has  been  definitely  established.  Their  constitution  still  offers 
a  fascinating  problem  for  the  organic  chemist. 

The  writer  favors  Tswett's  terminology  of  carotinoids  for  these  pig- 
ments. From  the  standpoint  of  phy  to  chemistry  there  is  definite 
evidence  for  the  existence  of  five  carotinoids,  with  indications  that 
several  others  also  occur.  When  it  was  discovered  that  certain  of  the 
carotinoids  occur  in  animals  it  was  believed  that  both  plants  and  ani- 
mals synthesize  these  pigments.  It  soon  became  apparent,  however, 
that  the  chromolipoids  found  in  the  higher  animals,  at  least  those 

7 


8  PREFACE 

which  have  been  identified  as  carotinoids,  are  in  reality  merely  derived 
from  the  food.  The  assumption  therefore  seems  justified  that  a  similar 
biological  relationship  exists  between  all  the  chromolipoids  of  plant 
and  animal  life;  in  other  words,  that  all  animal  chromolipoids  are 
derived  pigments  and  are  either  true  or  modified  carotinoids. 

The  writer  has  had  three  main  ideas  in  mind  in  preparing  this  mono- 
graph. First,  he  has  attempted  to  compile  a  thorough  history  of  the 
development  of  the  chemistry  of  the  plant  and  animal  chromolipoids. 
This  has  not  been  attempted  before  in  this  particular  field.  Second, 
he  has  tried  to  present  such  information  regarding  the  pigments  as 
would  be  useful  to  workers  who  desire  to  attack  the  many  interesting 
problems  in  this  branch  of  plant  and  animal  chromatology.  Third,  he 
has  made  an  effort  to  point  out  lines  of  research  which  might  prove 
attractive  to  those  interested  in  this  subject.  The  author  hopes  that 
he  has  had  a  reasonable  measure  of  success  in  his  efforts. 

For  the  convenience  of  readers  who  have  not  been  trained  in  sys- 
tematic nomenclature  the  scientific  name  of  the  individual  species  of 
plants  and  animals  in  which  carotinoids  occur  has  been  supplemented 
wherever  possible  by  the  common  name.  For  the  plants,  this  informa- 
tion has  been  drawn  largely  from  Bailey's  Cyclopedia  of  Horticulture^. 

It  may  be  of  interest  to  the  reader  to  know  that  the  carotinoid  pig- 
ments in  plants  and  animals  have  proved  to  be  of  some  practical 
importance.  The  uses  to  which  their  occurrence  in  animals  have  been 
put  are  reserved  for  discussion  in  Chapter  XI  of  the  monograph.  The 
occurrence  of  carotinoids  in  plants,  particularly  green  plants,  formed 
the  basis  for  the  construction  of  the  light  filters  used  by  the  American 
Army  during  the  late  war  for  the  detection  of  camouflaged  foliage. 
Natural  green  foliage  reflects  both  green  and  red  light,  due  to  the  fact 
that  the  chlorophylls  and  carotinoids  are  present  together  in  the  chloro- 
plastids.  The  visibility  of  the  rays  reflected  from  the  carotinoids  is 
so  low  in  the  presence  of  the  chlorophylls  which  are  present  in  five  to 
six  times  the  concentration  of  the  orange  and  red  pigments,  that  green 
color  only  appears  to  be  reflected.  However,  it  was  found  possible  to 
construct  a  light  filter  which  absorbed  practically  all  light  rays  except 
a  wide  band  in  the  red  at  about  700\i\i,  and  a  narrow  band,  with  low 
transmission  in  the  green  at  about  500[X[x,  so  that  natural  green  foliage 
viewed  through  this  filter  appeared  red,  while  camouflaged  foliage  on 
which  green  paint  only  was  used,  appeared  green. 

The  writer  has  encountered  so  much  difference  of  opinion  regarding 
the  correct  pronunciation  of  certain  words  which  are  used  very  fre- 


PREFACE  9 

quently  in  this  monograph  that  he  begs  to  suggest,  for  the  sake  of 
uniformity,  the  following  pronunciations  which  are  believed  to  be  in 
keeping  with  the  best  modern  English  usage. 

Carotin  =Kar'o-tin. 

Carotinoid         =:Kar'' 6  -  tin- oid'. 

Lipochrome       =Lip'o-kr6m. 

Chromolipoid    =  K  r  o  m  -  o  -  1  i  p'  o  i  d. 

Chromatogram  —  K  r  6  m''  -ato-gra  m'. 

In  conclusion  the  author  wishes  to  acknowledge  his  indebtedness  to 
his  colleague.  Dr.  R.  A.  Gortner,  for  many  helpful  criticisms  in  the 
preparation  of  the  manuscript;  to  Dr.  Josephine  E.  Tilden  of  the 
Department  of  Botany,  University  of  Minnesota,  for  classifying  the 
algsB  in  which  carotinoids  occur,  and  to  Mr.  Lloyd  A.  Jones,  of  the 
Eastman  Kodak  Co.,  for  information  regarding  the  light  filters  devised 
in  the  Eastman  Research  laboratories  during  the  war. 

St.  Paul,  Minn. 
July  1,  1922. 


CONTENTS 

PAGE 

Chaptek  I.     General  Distribution  of  Carotinoids.    The  Pigments 

Defined 13 

Luteins.  —  Lipochromes.  —  Lipoxanthins.  ■ —  Chromolipoids. 
— Carotinoids. — Non-carotinoid  plant  pigments. — Non-caro- 
tinoid  animal  pigments. 

Chapter  II.     Carotinoids  in  the  Phanerograms     .....       25 

The  pigments  of  the  carrot. — Carotinoids  in  other  roots. — 
Carotinoids  in  the  chloroplastids. — Separation  of  yellow  pig- 
ments from  chlorophyll. — Crystalline  carotinoids  from  chlo- 
roplastids.— Plurality  of  yellow  pigments  in  chloroplastids. 
— Carotinoids  in  etiolated  leaves. — Carotinoids  in  naturally 
yellow  leaves. — Carotinoids  in  yellow  autumn  leaves. — Caro- 
tinoids in  autumn  and  winter  reddening.— Carotinoids  in 
flowers. — Carotinoids  in  fruits. — Carotinoids  in  seeds  and 
grains. 

Chapter  III.    Carotinoids  in  the  Cryptogams 92 

Carotinoids  in  the  algae. — The  Phseophyceae. — Fucoxanthin. 
— The  Rhodophyceae. — The  Charales. — The  Chlorophyceae, 
— The  Baccilariae  (Diatomaceae) . — The  Peridinieae. — The 
Flagellata. — The  Myxophyceae  (Cyanophyceae) . — Carotin- 
oids in  the  fungi. — The  Basidiomycetes. — The  Ascomycetes. 
— The  Phycomycetes. — The  Myxomycetes. — The  Imperfects. 
— Carotinoids  in  Bacteria. 

Chapter  IV.     Carotinoids  in  the  Vertebrates 125 

Carotinoids  in  mammals. — Corpus  luteum. — Blood  serum. — 
Milk  fat. — 'Adipose  tissue. — Internal  organs. — Nerves. — Skin. 
— Carotinoids  in  birds. — Egg  yolk. — Body  tissues. — Retina. 
— Feathers. — Carotinoids  in  fishes. — Carotinoids  in  amphib- 
ians.— Carotinoids  in  reptiles. 

Chapter  V.     Carotinoids  in  Invertebrates 154 

Carotinoids  in  insects. — Lepidoptera. — -Rhynchota. — Coleop- 
tera. — Orthoptera. — Acerata. — Carotinoids  in  Crustacea. — 
Carotinoids  in  Echinoderms. — Carotinoids  in  molluscs. — 
— Carotinoids  in  worms. — Carotinoids  in  sponges. 

11 


12  CONTENTS 

PAGE 

Chapter  VI.     Chemical  Relations  between  Plant  and  Animal 

Carotinoids 173 

Egg  yolk  xanthophyll, — Corpus  luteum  carotin. — Crustacean 
carotin. 

Chapter  VII.    Biological  Relations  between  Plant  and  Animal 

Carotinoids 182 

Earlier  views  for  and  against  a  biological  relationship. — -Iso- 
lated facts  supporting  a  biological  relationship. — Experi- 
ments proving  a  biological  relationship. — Insects. — Cattle. — 
Fowls. — Man. — Distributions  of  carotinoids  among  different 
species. 

Chapter  VIII.    Methods  of  Isolation  of  Carotinoids      .     .     .     199 

Isolation  of  carotin. — From  carrots. — From  green  leaves. — 
From  animal  fat. — From  blood  serum. — Isolation  of  xan- 
thophylls. — From  green  leaves. — From  egg  yolk. — From 
blood  serum. — Isolation  of  lycopin.— Isolation  of  fucoxan- 
thin. — Isolation  of  rhodoxanthin. 

Chapter  IX.    General  Properties  and  Methods  of  Identification 

of  Carotinoids     . 218 

Properties  of  carotinoid  solutions. — Carotin. — ^Lycopin. — 
Xanthophylls.  —  Rhodoxanthin.  —  Fucoxanthin.  —  Proper- 
ties of  crystalline  carotinoids. — Carotin. — Other  pigmented 
hydrocarbons.  —  Xanthophyll.  —  Lycopin.  —  Fucoxanthin. 
— Methods  of  identification  in  biological  products. — Plant 
tissues. — Animal  tissues. 

Chapter  X.    Quantitative  Estimation  of  Carotinoids      .     .     .     248 

Estimation  of  carotin  and  xanthophyll. — Method  of  Arnaud. 
— Arnaud's  results. — Method  of  Monteverde  and  Lubimenko. 
— Monteverde's  results. — Methods  of  Willstatter  and  Stoll. — 
Results  by  Willstatter  and  Stoll's  method. — Estimation  of 
fucoxanthin. — Application  to  other  biological  materials. 

Chapter  XI.    Function  of  Carotinoids  in  Plants  and  Animals  .     262 

Possible  function  in  plants. — Possible  function  in  animals. — 
Possible  relations  to  vitamins. — Relation  between  yellow  pig- 
mentation and  fowls  and  egg  laying. — Possible  relation  be- 
tween yellow  pigmentation  of  cattle  and  milk  secretion. 

Bibliography 279 

Index  to  Authors 296 

Index  to  Subjects 300 


CAROTINOIDS  AND  RELATED 
PIGMENTS 

Chapter  I 

General  Distribution  of  Carotinoids.    The  Pigments 

Defined 

Red,  orange  and  yellow  pigments  which  can  be  extracted  from  tis- 
sues by  fat  solvents  are  found  abundantly  in  all  forms  of  living  mat- 
ter. In  the  plant  world  they  are  present  in  nearly  all  species  ranging 
from  bacteria,  the  lowest  forms  of  cryptogams,  to  the  dicotyledons, 
the  highest  forms  of  phanerogams.  Similarly  in  the  animal  kingdom 
we  find  yellow  to  reddish  pigments  in  all  forms  of  both  invertebrates 
and  vertebrates,  from  protozoa  to  man.  The  earliest  workers  in  both 
the  plant  and  animal  fields  naturally  based  the  classification  of  the 
pigments  on  simple  properties,  so  that  it  is  not  surprising  to  find  that 
many  names  have  been  proposed  for  what  is  obviously  the  same 
pigment. 

This  diversity  in  nomenclature  is  found  to  be  especially  true  among 
the  yellow  animal  pigments,  and  can  be  traced  in  most  instances  to 
slight  variations  in  certain  of  the  simple  properties  which  were  re- 
garded as  specific  for  various  types  of  pigment.  In  some  cases  these 
variations  were  due  to  the  fact  that  the  method  employed  for  the  iso- 
lation of  the  pigment  did  not  insure  its  freedom  from  other  pigments  of 
similar  but  not  identical  properties.  In  other  cases  the  variations  were 
due  to  the  examination  of  the  pigment  in  amorphous  condition  or  in 
solution,  without  reference  to  the  possible  effect  which  these  states 
might  exert  upon  the  particular  properties  being  studied.  Again,  there 
was  frequently  an  abundant  contamination  with  lipoid  impurities, 
which  are  invariably  separated  with  the  pigments  from  animal  tis- 
sues. Another,  still  more  important  cause  for  these  variations  was 
the  failure  to  protect  the  pigments  from  oxidation.  The  true  caro- 
tinoids, which  unquestionably  comprise  the  great  majority  of  yellow 

13 


14  CAROTINOIDS  AND  RELATED  PIGMENTS 

and  red  tinted  animal  pigments  included  under  the  older  term  lipo- 
chrome,  are  characterized  by  the  ease  with  which  they  oxidize  when  in 
solution  or  in  the  solid  state.  The  earlier  workers  did  not  recognize, 
however,  that  some  of  the  most  characteristic  properties  of  these  pig- 
ments are  subject  to  modification  even  in  the  earliest  stages  of  oxida- 
tion. This  is  particularly  true  of  the  color  reactions  with  various 
reagents,  and  the  spectroscopic  properties,  which  have  been  used  so 
widely,  and  many  times  exclusively,  as  the  basis  for  the  classification 
of  the  animal  chromolipoids. 

Confusion  in  terminology,  however,  has  not  been  confined  to  the 
animal  pigments.  The  chief  difficulty  regarding  the  plant  carotinoids 
has  been  the  proposal  of  names  already  in  use  for  pigments  of  obvi- 
ously different  composition  and  properties.  For  example,  the  name 
xanthophyll,  as  used  by  various  workers  in  the  field  of  plant  pigments, 
has  been  the  cause  of  so  much  confusion  in  the  nomenclature  as  to 
be  very  disconcerting  to  many  students  of  this  subject. 

It  is  not  surprising,  therefore,  that  certain  investigators  have 
attempted  to  bring  some  semblance  of  order  to  the  confusion  by  pro- 
posing one  name  to  cover  all  the  names  previously  proposed'  for  pig- 
ments of  like  or  similar  properties.  A  brief  history  of  these  attempts 
with  their  resulting  influence  on  the  nomenclature  of  plant  and  animal 
chromatology  may  prove  of  interest  at  this  point. 

Luteins 

The  first  attempt  to  bring  various  yellow  pigments  together  under 
one  name  is  found  in  Thudichum's  (1869)  classic  paper,  in  which  the 
yellow  pigments  found  in  many  tissues  of  both  vegetable  and  animal 
origin  are  grouped  under  the  name  "luteine,"  or  luteins.  The  name 
was  obviously  suggested  by  the  fact  that  the  characteristic  yellow  pig- 
ment of  the  corpus  luteum  on  the  ovaries  of  mammals,  especially  that 
of  the  cow,  is  one  of  the  representatives  of  the  "luteine"  pigments.  It 
is  doubtful  whether  Thudichum  was  familiar  with  the  work  of  Pic- 
colo and  Lieben  (1866),  who  had  crystallized  the  corpus  luteum  pig- 
ment a  few  years  previously  and  named  it  luteohamatoidin  or  hamo- 
lutein.  However,  Thudichum  mentioned  the  work  of  Holm  (1867), 
who  isolated  the  corpus  luteum  pigment  and  called  it  hamatoidin. 

Thudichum's  luteins  included,  besides  the  corpus  luteum  pigment, 
the  yellow  pigment  of  blood  serum,  adipose  tissue  and  butter,  and  the 
yellow  pigment  of  egg  yolk.     The  vegetable  pigments  in  the  lutein 


GENERAL  DISTRIBUTION  OF  CAROTIN OIDS  15 

group  included  the  pigment  of  yellow  maize,  and  annatto  seeds,  the 
pigment  of  the  carrot  root  and  of  yellow  leaves,  such  as  those  of  the 
Coleus,  and  the  pigments  which  characterize  the  stamens  and  petals  of 
many  flowers. 

The  basis  for  the  classification  of  these  pigments  into  one  group 
was:  (1)  their  common  solubility  in  alcohol,  ether  and  chloroform, 
and  in  albuminous  liquids  like  blood  serum;  (2)  the  fact  that  their 
solutions  showed  three  absorption  bands  in  the  blue,  indigo  and  violet 
region  of  the  spectrum;  (3)  the  fact  that  they  could  be  crystallized 
in  the  form  of  rhombic  plates;  (4)  certain  common  chemical  reactions, 
such  as  their  precipitation  by  mercuric  acetate  and  mercuric  nitrate, 
and  their  blue  color  reaction  with  nitric  acid,  when  the  pigments  were 
in  the  solid  state  or  in  solution  in  acetic  acid;  (6)  and  their  affinity 
for  albumin,  as  in  blood  serum  and  the  fluid  of  ovarian  cysts,  from 
which  the  pigments  are  extracted  with  difficulty. 

Thudichum's  classification  never  received  wide  adoption.  In  fact, 
the  luteins,  as  defined  by  Thudichum,  comprise  a  number  of  different 
pigments.  Moreover,  our  present  knowledge  regarding  practically  all 
the  pigments  which  were  included  in  this  classification  shows  that  cer- 
tain of  the  characteristic  lutein  properties  ^  are  specific  only  for  cer- 
tain individuals  of  the  group.  The  final  abandonment  of  this  classifi- 
cation appears  in  the  recent  application  of  the  name  lutein  by  Will- 
statter  and  Escher  (1912)  to  the  specific  crystalline  pigment  isolated 
by  them  from  the  yolks  of  hen's  eggs.  This  use  of  the  name  appears 
to  the  author  to  be  illogical  both  from  the  standpoint  of  function  and 
anatomy  as  well  as  on  other  biological  grounds.  The  name  lutein 
obviously  suggests  the  body  from  which  the  name  was  derived,  namely, 
the  corpus  luteum.  The  yolk  of  the  egg  of  the  oviparous  animal  is 
certainly  not  related  to  the  corpus  luteum  either  functionally  or  an- 
atomically. Moreover,  the  egg  yolk  pigment  has  been  demonstrated 
by  Palmer  (1915)  to  be  physiologically  as  well  as  chemically  identical 
with  at  least  one,  and  probably  a  group  of  the  plant  pigments  which 
are  known  as  xanthophylls.  Egg  yolk  xanthophyll  is,  in  fact,  a  true 
carotinoid,  or  mixture  of  carotinoids,  and  no  further  designation 
appears  necessary. 

» The  heretofore  inexplicable  property  of  being  precipitated  by  mercury  salts,  ascribed 
to  the  luteins  by  Thudichum,  becomes  clear  only  in  the  light  of  Palmer's  (1914  c)  obser- 
vations that  the  albumin  with  which  carotin  is  sometimes  associated  in  the  blood  serum 
of  animals  is  precipitated  by  mercury  salts.  It  is  also  possible  that  Thudichum 
observed  the  phenomenon  of  the  adsorption  of  carotin  by  mercury  salts  described  by 
Tswett  (1906  b). 


^^^(CKL  imnif^ 


°®TOM    COUt-*^ 


16  CAROTINOIDS  AND  RELATED  PIGMENTS 

Lipochromes 

,Krukenberg  (1882k,  1886),  a  number  of  years  after  Thudichum, 
proposed  the  name  lipochrome  to  cover  all  the  animal  and  plant  pig- 
ments which  had  previously  been  known  as  luteins,  carotins,  zoonery- 
thrin,  tetronerythrin,  chlorophan,  xanthophan  and  rhodophan.  The 
name  lipochrome  has  been  widely  adopted  and  due  to  the  very  broad 
basis  upon  which  the  name  was  founded  it  has  been  applied  to  numer- 
ous plant  and  animal  pigments  not  mentioned  by  Kruk.enberg,  or 
unknown  to  him.  Krukenberg  believed  that  all  the  pigments  which 
he  proposed  to  designate  as  lipochromes  were  associated  with  fat  in 
their  natural  state,  and  the  name  suggests  this  supposition  as  well  as 
their  capability  of  existing  in  association  with  fats  and  oils. 

It  was  obviously  the  intention  of  the  originator  of  the  name  lipo- 
chrome to  limit  it  to  pigments  of  yellow  or  reddish  tints,  but  the  name 
itself  is  applicable  to  pigments  of  many  other  colors,  such  as  chloro- 
phyll and  many  vegetable  dyes  of  various  colors,  which  have  a  marked 
affinity  for  fat.  Numerous  workers  object  to  the  use  of  the  name  lipo- 
chrome on  this  account.  Kohl  (1902a),  for  example,  in  his  extensive 
monograph  on  carotin,  objects  to  designating  this  pigment  as  a  lipo- 
chrome because  of  the  numerous  cases  in  which  it  is  known  to  occur 
free  from  fat,  and  also  because  he  believes  that  where  carotin  is 
actually  found  associated  with  fat  it  is  in  combination  with  the  fat 
and  not  merely  in  solution. 

The  particular  properties  by  which  Krukenberg  (1886)  proposed  to 
judge  whether  a  pigment  should  be  classified  as  a  lipochrome  are,  in 
general,  as  follows:  They  are  soluble  in  alcohols  (methyl,  ethyl  and 
amyl),  ether,  chloroform,  benzene,  carbon  disulfide,  petroleum  ether 
and  acetone;  in  the  solid  state  they  are  colored  blue-green  to  blue  by 
concentrated  sulphuric  and  nitric  acids  and  generally  blue-green  with 
iodine  in  potassium  iodide ;  they  show  two  and  sometimes  three  absorp- 
tion bands  in  the  blue  and  violet  region  of  the  spectrum;  they  are  not 
destroyed  on  boiling  with  alcoholic  caustic  alkalies;  in  the  solid  state 
they  are  greenish-yellow,  yellow,  orange  or  red,  and  their  solutions  are 
yellow;  they  are  very  sensitive  to  light  and  readily  bleach,  the 
bleached  pigments  being  similar  to  cholesterol. 

Subsequent  investigations  of  the  lipochromes,  using  the  class  char- 
acteristics defined  by  Krukenberg,  have  added  very  little  to  our  knowl- 
edge of  the  properties  of  these  pigments  considered  as  a  group,  but 
have  served  merely  to  define  more  closely  certain  of  the  criteria  enu- 


GENERAL  DISTRIBUTION  OF  CAROTINOIDS  17 

merated.  Krukenberg  believed  that  all  lipochromes  should  be  regarded 
as  composed  of  carbon,  hydrogen  and  oxygen,  and  free  from  nitrogen. 
At  the  present  time  hydrocarbons,  such  as  carotin  and  its  isomers, 
as  well  as  the  oxyhydrocarbons,  fulfill  all  the  characteristics  of  lipo- 
chromes. Probably  wider  use  has  been  made  of  the  color  reactions 
with  concentrated  sulphuric  and  nitric  acids  and  with  iodine  in  potas- 
sium iodide  than  any  of  the  other  class  characteristics  for  identifying 
pigments  as  lipochromes  although  many  studies  have  also  included 
spectroscopic  observations.  Unfortunately  the  color  reactions  and 
spectroscopic  properties  are  subject  to  greater  variation  than  any  of 
the  others  upon  which  the  classification  is  based.  The  result  of  the 
color  tests  as  well  as  the  quality  of  the  color  is  often  influenced 
strongly  by  admixture  with  foreign  substances,  and  this  is  apparently 
especially  true  for  the  reaction  with  iodine  in  potassium  iodide.  Simi- 
larly the  spectroscopic  absorption  properties  are  subject  to  wide  varia- 
tion as  to  the  position  of  the  bands  as  well  as  their  definiteness  by 
reason  of  admixture  with  impurities,  concentration  of  pigment,  and  the 
solvent  employed. 

Lvpoxanthins 

A  more  recent  attempt  than  Krukenberg's  to  bring  all  the  known 
plant  and  animal  pigments  with  like  properties  under  one  name  is 
that  of  Schrotter-Kristelli  (1895a),  who  proposed  to  group  together 
all  the  various  plant  and  animal  coloring  matters  which  had  previously 
been  known  as  etiolin,  chlorophyll  yellow,  xanthin,  anthoxanthin, 
lutein,  xanthophyll,  chrysophyll,  carotin,  phylloxanthin,  phycoxanthin, 
erythrophyll,  solanorubin,  lipoxanthin,  haematochrom,  chlororufin, 
bacteriopurpurin,  haemolutein,  vitellorubin  and  tetronerythrin.  He 
regarded  these  pigments  as  at  least  an  homologous  group,  if  not  com- 
pletely identical,  and  chose  the  name  lipoxanthin  as  the  most  suitable 
for  a  general  designation.  The  chief  characteristics  of  the  lipoxan- 
thins,  according  to  Schrotter-Kristelli,  are  their  affinity  for  fats,  their 
insolubility  in  water,  their  blue  color  reaction  with  concentrated  sul- 
phuric acid,  their  absorption  of  the  violet  end  of  the  spectrum,  their 
lack  of  fluorescence  when  in  solution,  and  their  ease  of  destruction  by 
light  and  oxygen.  Schrotter-Kristelli  believed  that  the  slight  dif- 
ferences in  the  spectroscopic  properties  of  the  various  pigments  were 
due  to  their  ease  of  destruction. 

According  to  this  author  lipoxanthins  have  been  demonstrated  to 
occur  in  all  green  leaves,  in  autumn  leaves,  in  many  flowers  and  fruits, 


18  CAROTINOIDS  AND  RELATED  PIGMENTS 

in  arils  and  roots,  in  algae  lichens,  fungi  and  bacteria;  among  ani- 
mals they  have  been  demonstrated  in  the  egg  yolk  of  the  sea-spider, 
in  the  retina  of  bird's  eyes,  in  insects,  such  as  ChrysomelidcB  and  Coc- 
cinellidce,  and  in  the  secretions  of  various  crustacege,  such  as  various 
kinds  of  Diaptoma,  and  Maia  squinado  as  well  as  in  still  lower  forms 
of  animal  life. 

The  lipoxanthins  are  thus  seen  to  be  a  more  or  less  indefinite  group 
of  pigments,  whose  classification  together  under  one  head  is  secured 
just  as  well  by  the  older  term  lipochrome,  which  no  doubt  explains 
why  the  proposed  term  never  received  wide  recognition. 

Chromolipoids 

As  our  knowledge  of  the  so-called  lipochromes  and  lipoxanthins  has 
been  extended  by  exhaustive  researches  regarding  the  various  indi- 
vidual representatives  from  both  plant  and  animal  sources  the  objec- 
tions which  have  been  raised  by  various  workers  to  terms  such  as 
lipochrome  and  lipoxanthin  seem  to  be  more  and  more  valid.  The 
botanists  have  been  the  first  to  definitely  break  away  from  the  old 
terminology  as  exemplified  by  the  citation  from  Kohl's  monograph. 
Czapek  (1913a)  proposes  to  meet  the  objections  to  the  name  lipo- 
chrome by  calling  the  pigments  chromolipoids.  His  point  of  view  is 
that  the  lipochromes,  at  least  in  plants,  are  to  be  classed  with  the 
lipoids  by  reason  of  their  many  fat-like  properties,  especially  solu- 
bility, and  also  because  of  their  widespread  occurrence  in  cells  in 
which  lipoids  are  known  to  exist.  Moreover,  the  lipochromes,  in  com- 
mon with  phosphatides  and  sterols,  absorb  oxygen  very  readily. 
Czapek's  terminology  has  much  in  its  favor,  in  the  opinion  of  the 
author.  It  is  at  least  preferable  from  many  standpoints  to  the  more 
or  less  misleading  term  lipochrome. 

Carotinoids 

Attempts  have  not  been  wanting  to  secure  uniformity  in  the  termi- 
nology of  the  yellow  plant  pigments.  The  first  yellow  plant  pigment 
to  be  isolated  in  crystalline  form  was  carotin,  the  pigment  of  the  root 
of  the  cultivated  carrot,  Daucus  carota.  At  one  time  the  name  caro- 
tin was  used  to  cover  all  the  plant  chromolipoids.  When  it  became 
known  that  differences  existed  between  many  of  the  so-called  caro- 
tins, the  name  was  changed  to  carotinen,  or  investigators  spoke  of  the 


GENERAL  DISTRIBUTION  OF  CAROTINOIDS  19 

"carotin  group."  The  discovery  that  carotin  itself  is  a  hydrocarbon 
led  to  the  adoption  of  the  name  "carotene,"  as  proposed  by  Arnaud 
(1886).  The  London  Chemical  Society  favors  the  spelling  "carrotene" 
for  the  hydrocarbon. 

Zopf  (1893a,  1895)  proposed  to  distinguish  between  two  groups  of 
carotins,  namely,  eucarotins  (true  carotins)  which  were  hydrocarbon 
in  nature  and  carotinins,  which  contained  oxygen  as  well,  and  formed 
compounds  with  the  alkali  and  alkaline  earth  metals.  It  should  be 
stated,  however,  that  Zopf  used  the  term  carotin  synonymously  with 
lipochrome  in  most  of  his  extensive  studies  of  the  pigments  of  the 
lower  forms  of  plants  and  animals.  His  eucarotins,  which  were  some- 
times called  yellow  carotins,  unquestionably  contained  representatives 
of  our  present  group  of  xanthophylls  whose  chemical  relation  to  caro- 
tin was  not  discovered  until  several  years  later.  The  carotinins  of 
Zopf  were  red  in  color.  The  belief  that  they  contained  oxygen  was 
based  on  the  fact  that  they  appeared  to  form  alkali  and  alkali  earth 
compounds.  Obviously  the  carotinins  are  not  related  to  the  oxygen- 
containing  xanthophylls,  as  known  at  the  present  time.  None  of  the 
true  carotinoids  so  far  isolated  in  pure,  crystalline  state  show  acid 
properties  like  the  so-called  carotinins.  The  nature  of  the  compounds 
which  the  latter  are  stated  to  form  with  sodium,  calcium  and  barium 
remains  to  be  determined,  as  well  as  their  true  relation  to  the  caro- 
tinoids. The  carotinins  appear  to  be  constituents  of  both  plants  and 
animals,  as  will  appear  from  a  fuller  account  of  them  given  in  Chap- 
ters III  and  V. 

Tswett  (1911a),  to  whose  ingenuity  we  owe  much  of  our  knowledge 
regarding  the  physico-chemical  properties  of  the  chromolipoids,  has 
proposed  the  term  "carotinoide"  for  the  various  chromolipoids  which 
are  chemically  and  generically  related  to  carotin.  He  would  desig- 
nate as  carotins  all  those  chromolipoids  whose  constitution  and  prop- 
erties show  themselves  to  be  hydrocarbons,  and  as  xanthophylls  all 
those  whose  constitution  and  properties  show  themselves  to  be  oxy- 
hydrocarbons  and  which  are  chemically,  as  well  as  generically,  related 
to  carotin. 

Tswett's  terminology  has  been  widely  adopted.  The  author  has' 
also  used  it  consistently  in  his  own  writings.  The  term  carotinoid  has 
the  objection,  however,  that  the  -oid  ending  is  derived  from  the  Greek 
eiBvs,  shape,  so  that  strictly  speaking  the  carotinoids  are  pigments 
which  resemble  carotin  in  form  or  structure  only.  As  yet  nothing 
definite  is  known  regarding  the  structure  of  the  carotinoids.     The 


20  CAROTINOIDS  AND  RELATED  PIGMENTS 

word  form  cannot  be  restricted  to  crystalline  form,  inasmuch  as  the 
crystalline  form  of  the  carotinoids  varies  widely  depending  upon  the 
solvent  from  which  they  separate.  As  will  be  pointed  out  later,  how- 
ever, the  carotinoids  must  of  necessity  be  closely  related  structurally. 
Their  close  chemical  relations  and  the  fact  that  they  are  invariably 
found  together  in  chlorophyllous  organs  support  this  view. 

Tswett's  terminology  has  given  promise  of  presenting  a  very  simple 
solution  of  the  difficulties  of  nomenclature  in  connection  with  the  vari- 
ous red  and  yellow  tinted  pigments  which  conform  to  the  properties 
of  the  so-called  lipochromes  so  widely  distributed  in  all  forms  of  plant 
life.  Unfortunately,  however,  Lubimenko  (1914,  1915,  1916)  has 
greatly  complicated  the  system  on  very  inadequate  evidence  by  using 
the  ending  -oid  for  a  group  of  pigments  which  he  believes  to  corre- 
spond to  each  of  the  definitely  known  carotinoids.  Thus,  Lubimenko 
speaks  not  only  of  carotin,  xanthophyll,  lycopin,  etc.,  but  of  caro- 
tinoids, xanthophylloids,  lycopinoids,  etc.,  as  well.  One  cannot  but 
express  the  opinion  that  our  knowledge  of  the  carotinoids  in  the  sense 
used  by  Tswett,  and  followed  in  this  monograph,  is  not  sufficiently 
extensive  to  warrant  a  belief  in  the  existence  of  numberless  interme- 
diate products.  As  a  matter  of  fact,  the  chemistry  of  the  specific  indi- 
viduals of  Lubimenko's  terminology,  namely  carotin,  lycopin,  xantho- 
phyll and  rhodoxanthin,  argues  against  the  existence  of  many  plant 
chromolipoids  of  the  nature  of  those  mentioned.  Certainly  in  view 
of  the  fact  that  there  is  every  evidence  to  believe  that  all  the  xan- 
thophylls  bear  the  simple  relation  to  carotin  that  is  expressed  in  their 
respective  formulae,  C^oHgg  and  C^oHgeOg,  it  seems  little  short  of  pre- 
posterous to  assume  the  existence  of  a  group  of  "carotinoids"  which 
are  oxidation  products  of  carotin  and  another  group  of  "xanthophyl- 
loids" which  are  reduction  products  of  xanthophyll. 

Tswett's  terminology,  therefore,  seems  entirely  adequate  for  our 
present  knowledge  of  the  chromolipoids  of  plant  origin.  If  the  chemi- 
cal and  physiological  relation  of  the  carotinoids  to  the  yellow  animal 
chromolipoids  of  the  tissues  and  fluids  of  the  higher  mammals  and 
man,  and  of  the  egg  yolk  and  bodies  of  oviparous  animals,  is  a  cri- 
terion of  similar  relations  throughout  the  entire  realm  of  the  animal 
kingdom,  then  Tswett's  terminology  is  equally  applicable  to  the 
yellow  and  red  tinted  chromolipoids  so  widely  distributed  in  all  forms 
of  animal  life.  The  probability  of  such  a  relationship  is,  in  fact,  the 
basis  of  the  present  monograph. 


GENERAL  DISTRIBUTION  OF  CAROTINOIDS  21 

Non-carotinoid  Plant  Pigments 

Carotinoids,  however,  are  not  the  only  yellow  and  orange  colored 
pigments  found  in  the  plant  and  animal  kingdoms,  which  fact  must 
not  be  lost  sight  of  in  the  examination  of  plant  and  animal  products 
for  the  presence  of  carotinoid  pigments. 

Although  all  plant  pigments  have  a  hydrocarbon  nucleus  there  are 
only  a  few  yellow  to  red  hydrocarbons  known  which  are  not  carotin- 
oids.  They  are  the  acenaphtylene  of  Behr  and  van  Dorp  (1873), 
Blumenthal  (1874)  and  Graebe  (1893),  the  di-biphenylenathene  of  de 
la  Harpe  and  van  Dorp  (1875)  and  Graebe  (1892),  the  fulvenes  of 
Thiele  (1900a),  the  cinnamylidenindene  of  Thiele  (1900b),  and  the 
rubicene  of  Pummerer  (1912).  Each  of  these  is  discussed  more  fully 
in  Chapter  IX,  in  connection  with  the  probable  constitution  of  carotin. 

Among  the  naturally  occurring  yellow  vegetable  pigments  which 
contain  carbon,  hydrogen  and  oxygen,  but  which  have  no  relation  to 
the  xanthophyll  group  of  carotinoids,  two  especially  well  defined 
groups  are  known,  namely,  the  xanthones  and  the  fiavones.  Five 
xanthones  are  known,  (1)  Cotoin,  CiaH^gO^,  (2)  Euxanthone, 
Ci3H6(0H)202,  (3)  Maclaurin,  Ci3Hg(OH)50,  (4)  Datiscetin,  or 
di-methyl-tetraoxyxanthone,  CigHigOg,  and  (5)  Gentisein,  C13H5- 
(0H)302.  The  structural  constitution  of  each  of  these  pigments  is 
known.  A  much  larger  number  of  fiavones  are  known,  all  of  which 
are  characterized  by  the  common  nucleus,  p-phenyl-benzo-y-pyrone. 
Many  of  the  natural  pigments  occur  as  glucosides  and  are  regarded 
as  the  chromogens  from  which  anthocyanins  are  derived  (Wheldale, 
1916).  Some  of  the  more  interesting  members  with  a  yellow  color 
are  (1)  Luteolin,  which  is  not  to  be  confused  with  the  carotinoid, 
Eteolin,  but  which  is  1,  2,  3,  4-tetra-oxyflavon;  and  (2)  Gossyptin,  the 
yellow  dye  in  the  yellow  flowers  of  the  Indian  cotton  {Gossypium 
herbaceum),  occurring  there  as  a  glucoside.  No  doubt  the  yellow 
color  of  cottonseed  meal  is  due  in  part  to  Gossyptin,  which  can  be 
extracted  from  cotton  flowers  with  hot  alcohol.  The  pure  pigment 
exists  as  glistening  yellow  needles. 

Besides  the  xanthones  and  flavones  other  yellow  pigments  are  found 
in  plant  parts,  among  which  may  be  mentioned  chrysophanic  acid,  a 
methyl  di-hydroxy  anthracene  whose  solution  in  alcohol,  ether,  ace- 
tone, benzene,  chloroform  or  petroleum  ether  will  dye  animal  tissues  a 
deep  yellow  color.  Of  special  interest  in  this  connection  is  the  yellow 
pigment  of  the  seeds  of  annatto   {Bixa  orellana),  called  bixin  or 


22  CAROTINOIDS  AND  RELATED  PIGMENTS 

annatto,  which  is  widely  used  for  the  artificial  coloring  of  butter  and 
cheese,  and  which  has  been  commonly  regarded  as  a  lipochrome  and 
was  included  by  Thudichum  among  the  luteins.  The  annatto  pig- 
ment does,  in  fact,  correspond  in  almost  every  particular  with  the 
class  characteristics  of  the  lipochromes.  It  is  not  entirely  unattacked 
by  alkalies,  however,  and  furthermore  is  decomposable  into  a  number 
of  well  known  substances,  such  as  m-xylene,  m-ethyl  toluene,  and  even 
palmitic  acid.  It  reduces  Fehling's  solution  even  in  the  cold.  Its 
constitution  is  unknown,  as  yet,  but  its  elementary  composition  cor- 
responds to  the  formula  CggHg^Os,  according  to  Etti  (1878),  or 
C29II34O5,  according  to  van  Hasselt  (1909).  It  is  thus  seen  that 
bixin,  while  corresponding  well  to  the  lipochrome  classification,  is  in 
no  sense  a  carotinoid.  Palmer  and  Kempster  (1919c)  have  shown  that 
the  annatto  pigment  has  no  effect  on  the  coloration  of  the  tissues 
when  fed  to  fowls. 

Other  vegetable  coloring  matters  of  a  yellow  color  giving  reactions 
in  some  cases  similar  to  carotinoids,  but  of  entirely  different  com- 
position, are  Crocin,  and  Crocitin,  flavones  which  are  found  in  the 
petals  and  pollen  grains  of  the  Indian  crocus  {Crocus  sativus),  which 
dissolve  in  concentrated  sulphuric  and  nitric  acids,  with  a  deep  blue 
color,  which  passes,  however,  into  a  brown  shade.  Another  yellow 
vegetable  dye  showing  a  like  reaction,  although  the  after  shade  with 
the  acid  reagents  is  yellow,  is  the  nyctanthin  which  Hill  and  Sikar 
(1907)  described  a  few  years  ago.  The  empirical  formula  for  this  dye 
CaoHaTO^,  has  an  interesting  resemblance  to  that  of  the  carotinoids, 
at  least  when  one  doubles  the  above  formula. 

The  yellow,  orange,  and  red  colors  seen  frequently  among  the  fungi 
of  the  lichen  and  mushroom  types  appear  to  be  due  in  many  cases  to 
pigments  of  a  nature  quite  different  from  the  carotinoids.  Chryso- 
phanic  acid,  mentioned  above,  sometimes  occurs  among  these  plants, 
as  well  as  many  other  like  coloring  matters  which  have  been  named 
of  Zopf  (1889b,  1892b,  1893b)  and  which  other  workers  have  found 
occurring  among  the  Basidiomycetes.  In  color  and  in  some  of  their 
solubility  properties  these  pigments  resemble  the  carotinoids,  and  cer- 
tain of  them  give  a  color  reaction  with  concentrated  sulphuric  acid 
which  is  not  unlike  that  regarded  as  characteristic  of  the  lipochromes. 

N on- carotinoid  Animal  Pigments 

Several  yellow  pigments  are  present  in  animal  tissues  and  fluids 
which  are  not  to  be  mistaken  for  carotinoids.     One  of  these,  whose 


GENERAL  DISTRIBUTION  OF  CAROTINOIDS  23 

constitution  is  unknown,  but  which  is  thought  to  be  derived  from  the 
blood  corpuscles,  is  hamatoidin,  a  yellow  crystallizable  pigment  found 
in  old  blood  exudates,  in  mummified  embryos,  and  sometimes  in  the 
urine  and  other  excreta.  First  described  and  named  by  Virchow 
(1847)  and  later  by  others,  its  origin  as  well  as  chemical  properties 
and  crystalline  form  have  been  recently  studied  anew  by  Neumann 
(1904,  1905).  Holm  (1867)  thought  the  corpus  luteum  pigment  was 
hamatoidin  in  his  early  study  of  this  pigment  which  has  since  been 
shown  to  be  carotin. 

Another  much  more  widespread  yellow  animal  pigment  with  cer- 
tain lipochrome  properties  is  the  bile  pigment  bilirubin.  There  is 
less  danger  of  confusing  it  with  carotinoids,  however,  save  with  respect 
to  its  color,  inasmuch  as  it  is  a  nitrogen  containing  substance  which 
readily  forms  salts  with  the  alkali  and  alkaline  earth  metals,  and  has 
many  other  properties  at  variance  with  those  of  the  carotinoids. 

Other  non-carotinoid  pigments  exist  in  animal  tissues,  but  which 
resemble  the  carotinoids  in  color  and  in  solubility  in  fat  solvents. 
Palmer  and  Kempster  (1919a)  have  recently  encountered  such  a  pig- 
ment in  the  carotinoid-free  egg  yolks  of  hens  raised  from  hatching  on 
rations  devoid  of  carotinoids,  the  eggs  being  produced  likewise  on 
xanthophyll-free  rations.  The  small  amount  of  pigment  found  in  the 
yolks  could  be  extracted  by  acetone,  but  hardly  at  all  by  ether,  was 
almost  entirely  saponifiable  and  failed  to  respond  to  characteristic 
xanthophyll  tests.  The  author  finds  that  a  similar  pigment  can  be 
extracted  from  the  carotinoid-free  and  apparently  colorless  "corpus 
luteum"  of  the  sow,  if  a  sufficient  number  of  these  organs  are  mac- 
erated and  extracted  with  fat  solvent.  These  cases  are  cited  in  order 
to  point  out  the  danger  in  assuming  that  all  animal  pigments  of  a 
yellow  color  are  carotinoid  in  nature.  Such  a  sweeping  conclusion 
cannot  be  justified. 

The  same  statement  can  also  be  made,  although  with  less  assur- 
ance, for  certain  red  pigments  which  appear  among  the  lower  animals 
and  birds.  These  pigments,  as  indicated,  are  red  in  the  solid  condition 
but  their  dilute  solutions  are  usually  yellow.  They  have  been  studied 
by  certain  of  the  older  investigators,  such  as  Kuhne,  Maly,  Kruken- 
berg,  MacMunn  and  Zopf  and  others,  and  have  received  various 
names  at  the  hands  of  these  authors,  such  as  rhodophan,  vitellorubin, 
crustaceorubin,  tetronerythrin,  lina-carotin  (from  the  Lina  species  of 
beetles  in  which  they  occur)  and  diaptomin.  The  pigments  are  strik- 
ingly similar  in  many  respects  to  the  carotinoids,  but  differ  from  them 


24  CAROTINOIDS  AND  RELATED  PIGMENTS 

in  showing  only  one  wide  absorption  band  at  F,  and  in  forming, 
according  to  the  statement  of  certain  of  their  investigators,  true  com- 
pounds with  sodium,  calcium  and  barium.  These  points  of  divergence 
from  the  carotinoids  should  be  examined  in  the  light  of  our  present 
knowledge  of  carotin  and  xanthophylls  before  it  can  be  stated  with 
assurance  that  these  pigments  are  distinct  substances.  They  all  cor- 
respond completely  to  the  class  characteristics  of  the  older  termi- 
nology of  lipochromes. 

Summary 

Red,  orange  and  yellow  pigments  which  have  certain  simple  prop- 
erties in  common  are  found  in  almost  all  forms  of  plants  and  animals. 

These  pigments  have  been  variously  classified  as  luteins,  lipo- 
chromes, lipoxanthins  and  chromolipoids. 

These  classifications  have  been  based  on  general  properties  rather 
than  on  composition  and  are  accordingly  subject  to  both  error  and 
criticism. 

As  a  general  class  term  the  name  chromolipoid  seems  to  conform 
more  nearly  to  present  conceptions  of  these  pigments  as  well  as  to 
more  common  usage  in  connections  with  substances  with  fat-like 
properties. 

Investigations  regarding  the  composition  of  the  chromolipoids  show 
that  a  large  majority  of  them  are  apparently  chemically  and  gener- 
ically  related  to  carotin,  a  specific  chromolipoid  widely  distributed 
in  plant  and  animal  tissues. 

It  seems  reasonable  to  believe,  therefore,  that  a  great  many  chromo- 
lipoids can  be  classified  more  specifically  as  carotinoids,  a  name  pro- 
posed for  them  by  Tswett  (1911a). 

Two  classes  of  carotinoids  are  recognized  in  Tswett's  definition; 
carotins,  whose  constitution  and  properties  show  them  to  be  hydro- 
carbons identical  or  isomeric  with  carotin;  and  xanthophylls,  whose 
constitution  and  properties  show  them  to  be  oxy-hydrocarbons  and 
which  are  chemically,  as  well  as  generically,  related  to  carotin. 

Carotinoids  are  not  the  only  yellow  to  red  colored  pigments  occur- 
ring in  plants  and  animals.  Many  of  these  non-carotinoids  resemble 
the  true  carotinoids  in  one  or  more  properties  and  some  even  in  com- 
position. The  reader  is  referred  to  the  text  for  the  detailed  discussion 
of  the  non-carotinoids  and  the  properties  which  they  have  in  common 
with  the  carotinoids  as  well  as  their  distinguishing  characteristics. 


Chapter  II 

Carotinoids  in  the  Phanerogams 

There  is  no  special  reason,  either  physiological  or  genetical,  for 
considering  the  carotinoids  in  the  phanerogams  and  cryptogams  sepa- 
rately, as  is  done  in  this  and  the  subsequent  chapter.  In  fact,  there 
appears  to  be  no  logical  reason  for  subdividing  the  plants  into  groups 
in  connection  with  the  distribution  of  carotinoids,  inasmuch  as  the 
pigments  appear  to  be  widely  distributed  in  all  forms,  both  chloro- 
phyllous  and  non-chlorophyllous,  from  bacteria  to  dicotyledons.  The 
subdivision,  then,  is  merely  one  of  convenience. 

The  Pigments  of  the  Carrot 

The  pigment  of  the  carrot  root  {Daucus  carota)  was  first  described 
by  Wachenroder  (1826),  nearly  100  years  ago,  and  called  carotin  by 
him.  This  serves  as  the  starting  point  of  our  knowledge  of  the  prop- 
erties, as  well  as  the  nomenclature  of  the  carotinoids,  and  this  pigment 
today  represents  our  most  typical  chromolipoid.  For  this  reason  the 
carrot  pigment  will  be  considered  first. 

Wachenroder  made  an  ether  extract  of  the  dried  macerated  roots, 
or  the  coagulum  obtained  on  heating  the  carrot  juice.  The  golden 
yellow  salve-like  residue  left  on  evaporation  of  the  solvent  was  shaken 
repeatedly  with  ammonia  to  separate  admixed  fatty  material,  dis- 
solved again  in  ether  and  the  ether  allowed  to  evaporate  slowly  with 
addition  of  small  amounts  of  alcohol.  Ruby-red  quadratic  crystals, 
imbedded  in  fatty  material,  were  obtained.  Wachenroder  described 
the  crystals  as  tasteless  and  odorless,  soluble  in  alcohol  and  ether, 
readily  soluble  in  fats  and  ethereal  oils,  but  insoluble  in  acetic  acid 
and  alkalis. 

Vauquelin  and  Bouchardat  (1830)  are  credited  with  the  next  study 
of  the  carrot  pigment,  but  it  was  a  number  of  years  before  Zeise  (1847) 
isolated  carotin  from  carrot  roots  in  quantity  sufficient  for  analysis. 
Zeise  discovered  the  ready  solubility  of  the  pigment  in  carbon  disul- 
fide with  its  characteristic  blood  red  color,  as  well  as  the  fact  that 

25 


26  CAROTINOIDS  AND  RELATED  PIGMENTS 

alcohol  when  added  to  the  concentrated  carbon  disulfide  solution  will 
throw  down  the  carotin  in  crystalline  form.  The  beautiful,  glistening, 
copper  colored  crystals  were  described  by  Zeise,  who  also  mentioned 
their  insolubility  in  alcohol  and  their  difficult  solubility  in  ether  and 
acetone.  The  crystals  melted  at  168°  (+)C.  Zeise  made  the  first 
analysis  of  carotin  and  ascribed  to  it  the  formula  CgHg.  He  was  thus 
the  first  to  show  the  hydrocarbon  nature  of  the  pigment,  but  due  to 
the  authority  of  the  next  investigator  (Husemann  (1861)),  this  fact 
was  not  proved  until  Arnaud  (1886)  made  his  careful  analyses  of  the 
carrot  pigment. 

Husemann  (1861)  pressed  the  juice  from  finely  grated  carrots  and 
then  added  weak  sulphuric  acid  to  the  diluted  juice,  following  Zeise's 
method,  throwing  down  a  coagulum  which  was  partially  dried  and 
then  extracted  with  hot  80  per  cent  methyl-alcohol.  The  residue  was 
then  dried  completely  and  extracted  with  carbon  disulfide.  Carotin 
crystals  were  thrown  out  of  the  concentrated  carbon  disulfide  solution 
by  addition  of  absolute  alcohol.  Husemann  purified  the  crystals 
merely  by  repeated  washing  on  a  filter  with  hot  80  per  cent  alcohol 
and  finally  with  absolute  alcohol.  Husemann  described  the  ruby-red 
color  and  velvety  appearance  of  the  carotin  crystals,  and  their  violet- 
like odor,  which  he  found  was  especially  noticeable  on  warming.  He 
noticed  the  bleaching  of  the  crystals  in  the  air  with  the  complete  re- 
versal of  solubility,  the  bleached  crystals  being  very  difficultly  soluble 
in  carbon  disulfide  and  benzine,  but  easily  soluble  in  alcohol  and  ether. 
Husemann  found  that  carotin  was  not  precipitated  by  metallic  salts 
but  he  observed  the  green  color  produced  by  adding  ferric  chloride  to 
an  alcoholic  solution  of  the  pigment.  Palmer  and  Thrun  (1916)  have 
recently  studied  the  reaction  of  ferric  chloride  on  the  carotinoids  and 
have  found  it  a  most  useful  test  for  confirming  the  presence  of  these 
pigments  in  oils  and  fats  and  in  various  extracts  of  plant  and  animal 
tissues.  \ji        1 

Husemann  was  the  first  to  show  the  unsaturated  nature  of  the 
carotin  molecule,  although  he  regarded  the  chlorine  and  iodine  deriva- 
tives which  he  made  as  substitution  products.  Husemann's  analyses 
led  him  to  propose  the  formula  CigHa^O  for  carotin  and  his  figures 
were  accepted  over  those  of  Zeise. 

Arnaud  (1886)  was  the  next  investigator  of  the  carrot  chromolipoid. 
He  isolated  the  pigment  from  600  kilograms  of  carrots  by  pressing 
the  juice  from  the  grated  roots,  adding  lead  acetate  to  the  juice,  drying 
the  precipitate  in  vacuum  and  extracting  it  with  carbon  disulfide.    The 


CAROTINOIDS  IN  THE  PHANEROGAMS  27 

dried  press  cake  was  extracted  similarly  and  the  carbon  disulfide  dis- 
tilled off  of  the  combined  extracts.  The  residue  was  washed  with  cold 
petroleum  ether  and  the  pigment  purified  by  crystallization  from  car- 
bon disulfide  with  absolute  alcohol  and  then  allowing  it  to  crystallize 
spontaneously  from  cold  petroleum  ether.  About  3  grams  of  crystals 
per  100  kilograms  of  carrots  were  obtained  in  this  way. 

Arnaud  found  the  bleaching  of  carotin  noticed  by  Husemann  to  be 
an  oxidation,  analyses  which  he  made  of  the  bleached  product  show- 
ing an  addition  of  21  per  cent  oxygen.  The  rapid  bleaching  of  caro- 
tin solutions  was  also  noticed;  and  Arnaud  pointed  out  the  influence 
of  this  fact  on  the  securing  of  pure  preparations  for  analysis. 
Arnaud's.  analyses  of  freshly  prepared  crystals  showed  an  average 
composition  of  88.67  per  cent  carbon  and  10.69  per  cent  hydrogen, 
definitely  proving  the  correctness  of  Zeise's  assertion  regarding  the 
hydrocarbon  nature  of  the  pigment.  This  investigator  was  also  the 
first  to  prepare  the  crystalline  iodine  derivative  of  carotin  by  adding 
i"odine  crystals  a  little  at  a  time  to  a  solution  of  carotin  in  anhydrous 
petroleum  ether,  maintaining  the  while  an  excess  of  carotin  in  the 
solution.  It  was  the  elementary  composition  of  this  product,  con- 
sidered together  with  the  composition  of  the  pure  carotin,  that  led 
Arnaud  to  ascribe  to  carotin  the  formula  CasHag,  and  to  the  iodine 
derivative  the  formula  CseHggla. 

Kohl  (1902b)  has  given  us  one  of  the  most  detailed  descriptions  of 
the  chemical  and  physical  properties  of  carotin.  His  analyses  of  the 
crystalline  pigment,  however,  gave  unsatisfactory  results,  as  did  also 
his  molecular  weight  determinations,  using  the  cryoscopic  method. 
He  therefore  accepted  Arnaud's  formula  as  representing  the  correct 
composition  of  carotin.  Certain  of  Kohl's  detailed  descriptions  of 
carotin  will  be  summarized  in  Chapter  IX,  where  the  physical  and 
chemical  properties  of  the  carotinoids  are  considered. 

Willstatter  and  Mieg  (1907)  definitely  settled  the  composition  of 
the  carrot  carotin  at  the  time  they  proved  its  identity  with  the  carotin 
of  the  chloroplastid.  Their  data  show  a  mean  ratio  of  C:H  of  1:1.406 
for  the  carrot  carotin,  for  which  the  simplest  formula  is  C5H7.  Molecu- 
lar weight  determinations  in  CHCI3  and  CS2,  using  the  ebulloscopic 
method,  show  an  average  of  536,  which  corresponds  exactly  with  the 
formula  (CsHt)^.  or  C4oH5e,  which  thus  appears  to  be  the  correct 
formula  for  carotin. 

Escher  (1909)  and  Willstatter  and  Escher  (1910)  have  confirmed 
these  results  completely.    Escher  furthermore  attempted  to  ascertain 


28  CAROTINOIDS  AND  RELATED  PIGMENTS 

the  structure  of  carotin  using  150  grams  of  the  pigment  isolated  from 
carrot  meal  by  extraction  with  petroleum  ether.  His  efforts  led  only 
to  the  production  of  amorphous  products,  all  of  high  molecular  weight. 
The  constitution  of  the  pigment  thus  remains  to  be  determined. 

Euler  and  Nordenson  (1908)  also  isolated  carotin  from  carrots  in 
quantity  sufficient  for  analysis.  Their  results  confirm  the  Willstatter 
formula.  The  purified  crystals  from  25  kilos  of  fresh  carrots  were 
found  to  contain  xanthophyll,  which  was  identified  by  the  color  of 
the  crystals  and  their  solubility  properties.  Palmer  and  Eckles 
(1914g)  have  also  shown  the  presence  of  xanthophyll  carotinoids  in 
the  carrot  root  by  the  Tswett  (1906c)  chromatographic  method  of 
analysis,  but  van  Wisselingh  (1915),  using  microchemical  crystalliza- 
tion methods,  did  not  observe  any  xanthophyll  crystals. 

It  appears  that  anthocyanins,  also,  may  accompany  carotinoids  in 
the  carrot  root.  Wittmack  (1904)  has  described  a  red  variety  of 
carrots  {Daucus  carota,  var.  Boissieri  Schweinfurth)  which  contains 
both  carotinoids  and  anthocyanin. 

Many  other  investigators  have  isolated  carotin  crystals  from  car- 
rots without,  however,  submitting  them  to  chemical  examination. 
According  to  Schimper  (1885)  and  Courchet  (1888)  carotin  exists  in 
the  carrot  tissue  in  crystalline  form.  Van  Wisselingh  (1915),  how- 
ever, has  shown  that  the  little  tubules  which  Schimper  and  Courchet 
observed  are  not  true  crystalline  forms.  The  author,  also,  has  never 
observed  any  but  granular  deposits  of  carotin  in  sections  of  the  fresh 
carrot  tissue. 

Carotinoids  in  Other  Roots 

Very  few  other  roots  have  been  examined  for  carotinoids  although 
several  which  are  widely  used  as  food  are  characterized  by  their 
yellow  color,  e.g.,  the  yellow  parsnip  root  {Pastinaca  sativa) ,  and  the 
sweet  potato  {Ipomoea  batatas) ,  especially  the  highly  colored  varieties 
grown  in  the  southern  part  of  the  United  States,  popularly  called 
Yams.    The  pigments  of  these  roots  should  be  examined. 

Formanek  (1900)  has  studied  the  pigment  of  the  red  beet  {Beta 
vulgaris) ,  and  believes  that  the  red  pigment  changes  into  a  yellow  one 
under  certain  conditions.  The  absorption  bands  of  the  latter  are  iden- 
tical in  -position  with  those  of  carotin.  Formanek's  red  pigment 
showed  only  one  absorption  band  in  the  yellow  part  of  the  spectrum 
and  is  undoubtedly  an  anthocyanin.  Its  apparent  transformation  into 
carotin  cannot  at  present  be  explained. 


CAROTINOIDS  IN  THE  PHANEROGAMS  29 

Lubimenko  (1914a)  has  examined  the  pigment  of  the  yellow  turnip 
root  (Brassica  Rapa  L.)  and  finds  that  it  contains  a  yellow  pigment 
soluble  in  95  per  cent  alcohol,  but  which  he  was  not  able  to  crystal- 
lize, and  also  a  pigment  which  closely  resembles  lycopin,  the  red  pig- 
ment of  the  tomato.  Spectroscopically  the  pigment  appears  to  be 
identical  with  lycopin  but  because  of  a  difference  in  the  relative  inten- 
sity of  the  bands  as  compared  with  lycopin,  and  a  greater  ease  of 
solubility  in  alcohol  and  concentrated  acetic  acid,  Lubimenko  pre- 
ferred to  call  the  pigment  a  lycopinoid,  a  term  which  the  author 
regards  as  very  unfortunate  in  view  of  the  more  generally  accepted 
use  of  the  terminology  -oid  as  applied  to  the  carotins  and  xanthophylls. 

It  seems  possible  that  the  pigment  of  the  related  variety  of  turnips, 
namely,  rutabaga  (Brassica  campestris  L.)  is  of  the  same  character. 
The  question  of  this  type  of  carotinoid  in  roots  deserves  confirmation 
and  further  study. 

Carotinoids  in  the  Chloroplastids 

The  tissues  of  all  chlorophyllous  plants  are  characterized  by  certain 
specialized  bodies,  probably  protein  in  nature,  of  microscopic  size, 
called  plastids.  In  early  stages  of  the  plant's  development  and  often 
in  the  subterranean  parts  of  the  plant  after  maturity  the  plastids  are 
colorless.  They  are  then  called  leucoplastids.  More  commonly  they 
develop  green  pigments,  chlorophylls,  when  the  plastids  are  called 
chloroplastids,  or  chlorophyll  granules.  The  chlorophylls  in  the 
chloroplastids  are  always  accompanied  by  carotinoids  of  both  types, 
namely,  carotin  and  xanthophylls. 

Investigations  regarding  these  yellow  chromolipoids  in  the  chloro- 
plastids apparently  did  not  begin  until  the  observation  of  Fremy 
(1860)  that  a  yellow  pigment  can  be  obtained  from  green  leaves  by 
allowing  strong  HCl  and  ether  to  act  upon  the  residue  from  the  alco- 
holic extract,  or  by  similar  treatment  of  the  precipitate  thrown  down 
from  the  alcoholic  leaf  extract -by  A1(0H)3.  In  this  procedure  the 
ether  took  on  a  yellow  color,  the  pigment  of  which  Fremy  called 
phylloxanthine,  leaving  a  blue  pigment,  which  he  called  phyllocyanine, 
in  the  aqueous  acid  layer.  Fremy  believed  that  his  phylloxanthin 
pre-existed  in  the  leaves. 

It  is  now  quite  certain  that  Fremy's  phylloxanthine  was  a  mixture 
of  some  of  the  natural  carotinoids  of  the  leaf  with  an  acid  decompo- 
sition product  of  chlorophyll,  a  view  which  was  expressed  first  by 
Stokes   (1864).     The  name  phylloxanthin  is,  in  fact,  at  present  re- 


30  CAROTINOIDS  AND  RELATED  PIGMENTS 

served  for  a  product  of  rather  indefinite  composition  which  results 
from  the  action  of  acid  on  chlorophyll  b  (Tswett,  1907,  1908b) . 

In  a  later  study  of  methods  of  isolation  of  phylloxanthine,  however, 
Fremy  (1865)  undoubtedly  obtained  much  more  valid  proof  of  the 
existence  of  yellow  pigments  associated  with  chlorophyll  although  he 
regarded  the  pigment  which  he  isolated  as  the  same  yellow  phylloxan- 
thine isolated  by  the  ether-HCl  method.  He  found  that  a  careful 
addition  of  Mg(0H)2,  or  A1(0H)3  to  alcoholic  chlorophyll  solutions 
carried  down  the  green  pigment  only,  leaving  the  yellow  pigment  in 
solution.  Ca(0H)2  and  Ba(0H)2  gave  similar  results,  but  the  best 
procedure  with  the  last  named  reagent  was  to  add  an  excess,  which 
threw  down  all  the  pigments,  from  which  the  phylloxanthine  (caroti- 
noids)  could  be  extracted  with  alcohol.  Especially  interesting  was 
Fremy 's  observation  that  when  his  chlorophyll  was  saponified  with 
strong  bases,  alcohol  took  up  the  yellow  "phylloxanthin"  from  the 
residue,  and  yellow  plate-formed  or  reddish  colored  prismatic  crystals, 
soluble  in  alcohol  and  ether,  could  be  obtained  from  this  solution. 
The  red  crystals  were  described  as  being  very  much  like  crystals  of 
potassium  dichromate,  and  having  a  strong  coloring  power.  It  would 
appear  as  though  Fremy  succeeded  in  obtaining  for  the  first  time 
crystals  of  carotin,  and  possibly  xanthophyll  also,  from  green  plants. 

Fremy 's  observations  precipitated  a  lively  interest  in  the  subject  of 
yellow  pigments  in  the  chloroplastids  which  resulted  in  a  number  of 
investigations  during  the  succeeding  years,  some  quite  independent  of 
the  others.  These  investigations  seem  to  fall  quite  naturally  into 
several  groups.  The  first  of  these  was  a  series  of  studies  confirming 
the  presence  of  yellow  substances  accompanying  chlorophyll  through 
the  development  of  suitable  methods  for  their  separation. 


Separation  of  Yellow  Pigments  from  Chlorophyll 

Stokes  (1864a)  is  to  be  credited  with  first  discovering  a  method  for 
separating  the  actual  yellow  pigments  accompanying  chlorophyll  and 
for  recognizing  the  existence  of  distinct  green  and  yellow  constituents 
in  the  plastids.  This  investigator  states,  "I  find  the  chlorophyll  of 
land-plants  to  be  a  mixture  of  four  substances,  two  green  and  two 
yellow,  all  possessing  distinctive  properties";  and  later  referring  to 
phylloxanthine  he  states,  "When  prepared  by  removing  the  green 
bodies  by  A1(0H)3  and  a  little  water,  it  (phylloxanthin)  is  mainly 
one  of  the  yellow  bodies,  but  when  prepared  by  HCl  and  ether,  it  is  a 


CAROTINOIDS  IN  THE  PHANEROGAMS  31 

mixture  of  the  same  yellow  body  (partly,  it  may  be,  decomposed)  with 
the  product  of  decomposition  by  acids  of  the  second  green  body." 
Stokes  never  published  his  method  of  separation  in  detail  but  he  gives 
a  hint  of  its  character  in  a  paper  in  another  publication  (1864b),  in 
which  he  states  in  a  discussion  of  the  advantages  of  a  partition  between 
solvents  for  the  separation  of  various  substances,  "Bisulphide  of  car- 
bon in  conjunction  with  alcohol  enabled  the  lecturer  to  disentangle  the 
colored  substances  which  are  mixed  together  in  the  green  coloring 
matter  of  leaves." 

Stokes  was  not  the  only  one  of  the  earlier  investigators  to  express 
the  belief  that  Fremy's  pigments  were  decomposition  products.  Filhol 
(1865)  also  reached  this  conclusion,  as  did  Askenasy  (1867).  Filhol 
(1868)  a  little  later  noticed  that  it  is  possible  to  remove  the  green 
constituent  of  crude  alcoholic  chlorophyll  solutions  by  treating  them 
with  animal  charcoal  insufficient  to  completely  decolorize  the  solution. 
A  yellow  colored  solution  remained  on  filtering  off  the  bone-black,  the 
color  of  which  Filhol  believed  to  be  due  to  a  pre-existing  pigment  or 
pigments  associated  with  the  green  one.  C.  A.  Schunck  (1901),  many 
years  later,  employed  this  method  of  obtaining  his  xanthophyll  group 
of  pigments  free  from  chlorophyll.  Schunck's  "xanthophylls"  included 
carotin  also,  so  that  Filhol's  observation  was  in  reality  of  much  more 
importance  than  he  realized. 

Timiriazeff  (1871),  studying  Fremy's  phylloxanthin,  also  found  that 
alcohol  alone  would  extract  the  yellow  pigment  from  the  barium  com- 
pound thrown  down  from  alcoholic  leaf  extracts  by  an  excess  of 
Ba(0H)2.  He  preferred  to  call  the  yellow  pigment  xanthophyll,  the 
name  previously  employed  by  Berzelius  (1837a) — from  xavSog^  yellow 
and  ^vXXov,  leaf — for  the  yellow  pigment  which  he  extracted  from  the 
yellow  autumn  leaves  of  the  pear  tree  [Pyrus  communis).  Sorby 
(1871b)  employed  the  same  term  for  a  group  of  yellow  and  orange  pig- 
ments which,  with  chlorophyll,  he  believed  caused  the  green  color  of 
leaves,  and  were  represented  as  types  by  pigments  which  could  be 
extracted  from  carrots  by  CSg. 

Notwithstanding  the  previous  observations  of  Fremy,  Stokes,  Filhol, 
Timiriazeff  and  Sorby,  credit  is  given  in  most  quarters  to  Gregor 
Kraus  (1872a)  for  making  the  first  actual  separation  of  the  pigments 
of  leaf  extracts  from  one  another.  Kraus'  method  is  frequently  re- 
ferred to  as  an  ''ausschiittlungs"  method,  for  he  shook  the  green 
alcoholic  leaf  extracts  with  benzene  and  observed  that  the  benzene 
had  extracted  the  green  pigment  leaving  the  alcohol  -layer  a  beautiful 


32  CAROTINOIDS  AND  RELATED  PIGMENTS 

yellow.  Kraus  named  the  green  pigment  cyanophyll  and  the  yellow 
pigment  xanthophyll.  From  our  present  knowledge  of  the  relative 
solubility  properties  of  the  carotinoids  which  have  been  built  very 
largely  upon  Kraus'  observations,  it  is  obvious  that  his  xanthophyll 
pigment  was  composed  almost  entirely  of  the  xanthophyll  carotinoids, 
although  traces  of  carotin  may  have  been  present  also.  Kraus  found 
that  the  absorption  bands  of  his  xanthophyll  fraction  in  the  blue  part 
of  the  spectrum  corresponded  with  Bands  V  and  VI  of  the  leaf  extract. 
The  residue  from  Kraus'  xanthophyll  fraction  gave  a  deep  blue  colora- 
tion with  concentrated  sulphuric  acid,  and  the  xanthophyll  solution 
itself  bleached  very  quickly  in  the  sunlight. 

Several  studies  of  the  leaf  pigment,  using  the  Kraus  procedure,  soon 
followed.    Konrad  (1872)  observed  that  the  separation  of  xanthophyll 
from  chlorophyll  by  benzene  was  effective  only  when  an  alcohol  of 
about  70  per  cent  (by  volume)  strength  was  employed.    Treub  (1874) 
substantiated  the  necessity  of  using  weaker  alcohol  for  the  benzene 
separation,  and  found  that  CSg  was  effective  when  the  chlorophyll 
extract  was  in  strong  alcohol.    Cempert  (1872)  found  that  linseed  oil 
could  be  used  in  place  of  benzene  and  Wiesner  (1874a,  b)  found  that 
a  number  of  vegetable  oils   (linseed,  walnut,  poppyseed,  olive)   and 
ethereal  oils  (turpentine  oil,  rosemary  oil,  oil  of  wintergreen)  and  also 
carbon  disulfide  could  be  employed.    Wiesner  used  85  per  cent  (by 
volume)   alcohol  and  benzene,  boiling  at  92-94°  C,  for  the  regular 
Kraus  separation.    Equally  good  results  were  obtained  with  toluene 
and  xylene,  or  mixtures  of  these  with  benzene.    Wiesner  even  suc- 
ceeded in  shaking  the  green  chlorophyll  out  of  alcoholic  leaf  extracts 
with  dilute  egg  albumin.     Especially  important  was  his  observation 
that  dilute  ammonia  or  caustic  alkali  solutions  acting  on  the  residues 
from  alcoholic  leaf  extracts  would  take  up  most  of  the  yellow  color 
leaving  behind  the  chlorophyll,  mixed  with  a  little  xanthophyll.    This 
observation,  pointing  to  the  resistance  of  the  yellow  chromolipoids  to 
alkalis  and  the  attacking  of  chlorophyll  by  the  same  reagents,  was 
later  developed  by  Hansen  (1884a)  and  is  still  widely  used  for  the 
separation   of  carotinoids  from  chlorophyll.     Hansen   boiled  young 
wheat  plants  in  water  for  one-half  hour,  dried  the  product,  extracted 
with  cold  96  per  cent  alcohol  in  the  dark,  concentrated  the  extract  to 
one-eighth  volume,  saponified  with  NaOH,  diluted  the  soap  solution 
with  water,  and  salted  out  the  soap  with  NaCl.    The  green  soap  thus 
obtained  was  extracted  with  petroleum  ether,  which  gave  a  yellow 
extract  which  Hansen  called  "chlorophyll  gelb,"  the  residue  being 


CAROTINOIDS  IN  THE  PHANEROGAMS  33 

called  "chlorophyll  griin."  Carl  Kraus  (1875)  observed  the  same 
facts  when  he  found  that  benzene  extracts  a  yellow  color  from  alco- 
holic leaf  extracts  made  strongly  alkaline  with  KOH.  He  called  the 
yellow  pigment  xanthin  and  regarded  it  as  a  decomposition  product 
of.  G.  Kraus'  xanthophyll. 

Possibly  following  the  hint  given  by  Stokes  (1864b),  Sorby  (1873) 
developed  a  separation  method  for  the  yellow  and  green  constituents 
of  a  number  of  types  of  plants  using  alcohol  and  carbon  disulfide. 
Sorby  named  five  members  of  a  "xanthophyll"  group  of  yellow  pig- 
ments as  well  as  two  chlorophylls,  but  pure  pigments  could  not  have 
been  obtained  in  most  cases,  since  the  methods  which  he  employed  will 
not  give  a  true  separation  of  the  various  carotinoids  of  the  chloro- 
plastids.  Of  the  various  pigments  named  by  Sorby  the  "yellow 
chlorophyll"  is  obviously  a  xanthophyll  mixed  with  some  chlorophyll, 
and  the  "orange  xanthophyll"  is  for  the  most  part  carotin.  Sorby's 
"xanthophyll"  and  "yellow  xanthophyll"  are  the  only  true  xantho- 
phylls,  the  former  being  in  all  probability  a  mixture  of  what  are  now 
called  a  and  a'  xanthophyll,  while  the  "yellow  xanthophyll"  appar- 
ently consisted  almost  wholly  of  our  present  (3  xanthophyll,  which  is 
characterized,  as  Sorby  found  for  it,  by  the  development  of  a  blue 
color  when  its  alcoholic  solution  is  treated  with  HCl. 


Crystalline  Carotinoids  from  Chloro'plastids 

The  second  group  of  studies  relating  to  the  yellow  pigment  in  the 
chloroplastids  deals  with  isolation  of  crystals  of  carotinoids,  and  ter- 
minated with  the  isolation  and  analysis  of  one  of  the  pigments  and 
the  discovery  of  its  identity  with  the  carotin  of  carrots.  The  various 
studies  were,  for  the  most  part,  independent  of  each  other  and  accord- 
ingly resulted  in  the  proposal  of  several  different  names  for  the 
chloroplastid  chromolipoids. 

Fremy's  (1865)  observations  regarding  crystalline  chromolipoids 
have  already  been  mentioned.  He  apparently  regarded  the  crystals 
as  related  to  his  phylloxanthin,  as  no  special  name  was  proposed  by 
him  far  the  crystalline  pigment.  Hartsen  (1873a),  however,  several 
years  later,  observed  golden-red  crystals  in  the  deposit  from  the  spon- 
taneous evaporation  of  an  alcoholic  extract  from  green  leaves.  He 
called  the  pigment  chrysophyll,  a  name  previously  applied  by  Sorby 
(1871b)  to  a  group  of  water-soluble  pigments  from  autumn  foliage, 
and  later   (1875)   described  a  method  for  purifying  the  crystals  by 


34  CAROTINOIDS  AND  RELATED  PIGMENTS 

washing  away  the  fat  and  chlorophyll  with  petroleum  ether,  taking 
up  the  pigment  in  alcohol  and  recrystallizing.  Hartsen  regarded  his 
chrysophyll  as  probably  identical  with  xanthophyll  (G.  Kraus)  and 
as  existing  together  with  chlorophyll  in  the  leaf.  According  to  Will- 
statter  and  Mieg  (1907)  Hartsen's  chrysophyll  was  probably  a  xan- 
thophyll in  the  sense  that  this  name  is  used  at  the  present  time,  but 
most  writers  have  regarded  it  as  identical  with  carotin.  Bougarel 
(1877),  a  little  later,  isolated  red  crystals  from  the  alcoholic  extract 
of  peach  and  sycamore-tree  leaves.  He  described  the  insolubility  of 
the  red,  green  reflecting  crystals  in  alcohol  and  ether,  and  their  solu- 
bility in  chloroform,  benzene  and  carbon  disulfide,  as  well  as  the  rose 
color  of  the  solution  in  the  last  named  solvent.  Notwithstanding  his 
familiarity  with  Hartsen's  chrysophyll,  which  he  mentions,  Bougarel 
regarded  his  pigment  crystals  as  a  new  substance  and  unfortunately 
proposed  the  name  erythrophyll  for  it,  which  had  already  been  given 
by  Berzelius  (1837b)  many  years  before  for  the  red,  alcohol  soluble 
pigment  which  he  isolated  from  red  cherries  {Prunus  cerasus),  black 
Johannis  berries  {Rihus  nigrum)  and  the  red  autumn  leaves  of  vari- 
ous plants,  and  which  was  also  used  by  Sorby  (1871,  1873)  for  a  group 
of  water-soluble  pigments.  The  erythrophyll  of  Bougarel  is  unques- 
tionably to  be  regarded  as  carotin. 

Dippel  (1878)  made  a  careful  study  of  the  absorption  spectra  of 
G.  Kraus'  xanthophyll  and  cyanophyll  and  the  products  of  the  action 
of  KOH  and  acid  on  the  pigments  prepared  according  to  the  Kraus 
method.  He  found  that  yellow  pigments  could  be  prepared  in  each 
case  but  that  the  absorption  spectrum  of  the  yellow  pigment  from 
the  acid  treatment  of  cyanophyll  was  entirely  different  from  the 
spectra  of  the  yellow  pigment  from  the  alkali  treatment  of  both 
xanthophyll  and  cyanophyll.  Dippel  proposed  the  name  xanthin 
(compare  C.  Kraus  (1875))  for  the  yellow  pigment  obtained  from 
Kraus'  xanthophyll  and  cyanophyll  on  treatment  with  alkali  and 
extracting  with  alcohol,  and  regarded  it  as  the  true  yellow  constituent 
of  chlorophyll.  The  absorption  bands  of  Dippel's  xanthin  obtained 
by  alkali  treatment  of  Kraus'  benzene-cyanophyll  layer  lay  at  490- 
456pi[x  and  455-435[i|i,  while  the  bands  of  Kraus'  xanthophyll,  as 
measured  by  Dippel,  lay  at  483-460[i[x  and  446-433[X[i.  These  meas- 
urements correspond  almost  exactly  with  those  of  carotin  and  xan- 
thophyll, respectively,  as  known  at  the  present  time.  Dippel's  xanthin 
is  to  be  regarded,  therefore,  as  composed  of  carotin  for  the  most  part. 
Borodin  (1883)  made  one  of  the  most  striking  contributions  to  our 


CAROflNOWS  IN  THE  PHANEROGAMS  35 

knowledge  of  crystalline  carotinoids  accompanying  chlorophyll  in  the 
chloroplastids.    Two  groups  of  pigments  were  described  by  him,  one 
characterized  by  their  slight  solubility  in  alcohol  and  great  solubility 
in  benzine,  corresponding  with  one  of  the  properties  of  carotin,  the 
other  group  being  characterized  by  their  ease  of  solubility  in  alcohol 
and  their  slight  solubility  in  benzine,  which  corresponds  with  one  of 
the  most  distinguishing  properties  of  the  xanthophylls.     No  names 
were  proposed  by  Borodin  for  his  crystalline  pigments  but  he  described 
in  detail  the  crystal  forms  and  certain  properties  of  two  carotins  and 
two  xanthophylls.     One  of  the  carotins  formed  orange-red  rhombic 
crystals  (he  obtained  these  from  the  alcoholic  extract  of  Spirogyra) 
and  the  other  bright  yellow  needles  with  a  strong  violet  or  rose-red 
nuance.     Of  the  two  xanthophylls  one  formed  straw  yellow,  ribbon- 
like scales  or  dark  brown,  crooked,  branching  rods,  and  the  other 
golden-yellow  "navikeln,"  an  English  synonym  for  which  the  author 
has  not  been  able  to  find.    The  latter  were  observed  especially  clearly 
by  Borodin  in  extracts  from  parsley  {Petroseiinum  sativum).    Borodin 
regarded  the  red  and  violet  tinted,  benzine-soluble  group  as  widely 
distributed  in  all  chlorophyllous  plants,  the  red  forms  being  identical 
with  Bougarel's  erythrophyll.     The  alcohol  soluble  forms  were  not 
regarded  by  Borodin  as  being  so  widely  distributed,  especially  the 
pigment  forming  the  golden-yellow  ''navikeln."    With  the  exception 
of    the  red  rhombic-formed   crystals   in   the   benzine-soluble    group 
Borodin's  crystal   forms   do  not  correspond  with   the   carotins   and 
xanthophylls  which  have  since  been  isolated  in  pure  form  by  various 
investigators  so  it  is  not  known  whether  they  represent  forms  which 
were   modified  by   the   solvents  employed   or   isomeric    carotin   and 
xanthophyll  carotinoids  not  yet  isolated  in  quantity.    The  latter  pos- 
sibility is  not  to  be  disregarded  in  view  of  the  various  yellow  chromo- 
lipoids  which  are  revealed  in  chloroplastids  using  Tswett's    (1906c) 
chromotographic  analytical  procedure. 

Guignet  (1885)  observed  orange  crystalline  material  from  extracts 
obtained  by  a  method  similar  to  that  used  by  Dippel  for  xanthin. 
Alcoholic  leaf  extract  in  strong  alcohol  was  agitated  with  one-tenth 
its  volume  of  petroleum  ether  (Sachsse  (1877)  introduced  the  use 
of  petroleum  ether-  instead  of  benzene  in  the  Kraus  separation)  and 
the  green  petroleum  ether  extract  agitated  with  a  solution  of  NaOH 
in  95  per  cent  alcohol,  leaving  a  yellow  solution  from  which  the  crys- 
talline material  was  obtained.  The  pigment  was  no  doubt  carotin 
although  no  name  was  proposed  for  it  by  Guignet. 


36  CAROTINOIDS  AND  RELATED  PIGMENTS 

It  remained  for  Arnaud    (1885),  however,  to  first  recognize  and 
prove  the  identity  of  the  orange-red  crystals  apparently  first  observed 
by  Fremy  (1865)  and  later  called  chrysophyll,  erythrophyll,  xanthin, 
etc.,  with  the  carotin  from  carrots,  which  had  been  known  and  studied 
for  60  years.    Arnaud  (1885)  first  observed  the  identity  in  form  and 
properties   of  carotin  which  he  isolated   from   carrots   and  the  red 
rhombic  crystals  which  he  isolated  from  spinach  leaves   {Spinachia 
oleracea  and  glabra),  mulberry  leaves    {Morus  alba),  the  leaves  of 
peach  (Persica  vulgaris)  and  sycamore  [Acer  pseudoplatanus)  trees, 
and  the  leaves  of  the  English  ivy  vine  {Hedera  helix) ,  as  well  as  from 
pumpkins  {Cucurbita  pepo).    In  a  succeeding  paper  Arnaud  (1886) 
proved  this  identity  by  his  analyses  of  the  crystals  obtained  from 
carrots,  to  which  reference  has  already  been  made,  the  results  leading 
to  the  proposal  of  the  formula  CasHgg  for  the  pigment.    Arnaud  did 
not  make  any  analyses  of  the  apparently  identical  crystals  which  he 
obtained  from  leaves,  so  that  strictly  speaking  the  final  proof  of  the 
identity  of  the  crystals  was  not  furnished  until  Willstatter  and  Mieg 
performed  their  comparative  analysis  many  years  later  (1907).    How- 
ever, following  Arnaud,  investigators  with  few  exceptions  adopted  his 
terminology  and  called  the  red  crystalline  pigment  carotin  which  could 
be  isolated  so  generally  from  chlorophyll  forming  plants,  as  well  as 
many  fruits  and  seeds,  and  from  cryptogamic  forms.     Immendorff 
(1889),  in  fact,  soon  after  Arnaud's  work,  isolated  carotin  from  barley 
and  rye  leaves  and  submitted  the  crystalline  pigment  to  analysis.    His 
data  corresponded  best  with  Zeise's  older  formula,  CgHg,  but  he  pre- 
ferred to  accept  the  Arnaud  formula  because  it  appeared  to  be  sub- 
stantiated by  Arnaud's  analysis  of  the  iodine  derivative  of  carotin. 
Willstatter  and  Mieg,  however,  starting  with  100  kilos  of  dried 
nettle  (Urtica)  leaves,  isolated  carotin  in  sufiicient  quantity  to  estab- 
lish  for  it  the   correct  formula,   C^oHgg.     Their   analyses  gave  the 
average  composition  of  89.29  per  cent  carbon  and  10.53  per  cent  hydro- 
gen as  compared  with  the  theoretical  values  89.48  and  10.52  per  cent 
carbon  and  hydrogen,  respectively. 

Plurality  of  Yellow  Pigments  in  the  Chloroplastids 

The  next  group  of  investigations  dealing  with  the  yellow  pigments 
of  the  chloroplastids  had  to  do  with  the  question  whether  more  than 
one  yellow  pigment  is  a  constant  accompaniment  of  the  chlorophyll. 
This  question  brings  us  up  to  the  present  time  for  notwithstanding  the 


CAROTINOIDS  IN  THE  PHANEROGAMS  37 

fact  that  two  crystalline  carotinoids  in  the  xanthophyll  group  have 
now  been  isolated  from  plant  forms  and  their  composition  determined, 
the  isolation  of  other  known  members  of  this  group  of  pigments  still 
remains  to  be  carried  out  and  their  composition  and  relation  to  the 
known  members  determined. 

Stokes  (1864a)  is  to  be  credited  with  the  first  suggestion  of  the 
presence  of  more  than  one  yellow  pigment  in  chloroplastids,  but  in 
spite  of  the  various  yellow  pigments  isolated  by  Sorby  (1873),  using 
Stokes'  carbon  disulfide  procedure,  this  method  could  not  have  led  to 
a  true  isolation  of  the  various  members  of  the  carotinoid  pigments 
which  are  recognized  today.  The  observation  of  Dippel  (1878)  that 
besides  Kraus'  xanthophyll  a  yellow  pigment  accompanied  the  cyano- 
phyll  in  the  petroleum  ether  layer,  could  have  led  to  the  discovery  of 
the  actual  existence  of  two  groups  of  carotinoids.  Borodin  (1883), 
however,  first  demonstrated  the  existence  of  more  than  one  yellow 
chromolipoid  when  he  obtained  various  forms  of  crystals  from  green 
plants.  As  already  pointed  out,  these  crystals  naturally  fell  into 
two  groups  according  to  their  solubility  properties,  one  group,  to 
which  Borodin  recognized  the  erythrophyll  (carotin)  of  Bougarel 
belonged,  being  very  soluble  in  benzine  (petroleum  ether)  and  dif- 
ficultly soluble  in  alcohol,  and  the  other  group  being  easily  soluble  in 
alcohol  but  dissolving  with  difficulty  in  benzine.  These  observations 
of  Borodin's  are  the  basis  of  the  classification  of  the  carotinoids  which 
prevail  at  the  present  time,  considerably  extended  and  supported,  of 
course,  by  other  chemical  and  physical  properties;  they  also  furnish 
the  basis  for  the  separation  of  the  carotinoids  into  the  two  groups  now 
recognized,  namely,  the  carotin  and  xanthophyll  groups.  This  sys- 
tem of  classification  is  the  only  logical  one,  as  has  been  pointed  out  by 
Tswett  and  proven  by  the  chemical  analyses  of  members  of  each  group 
by  Willstatter  and  his  co-workers.  Before  reviewing  the  history  and 
evidence  in  favor  of  this  classification  and  the  proof  of  the  existence 
of  individuals  in  the  group,  it  should  be  stated  that  the  plurality  of 
yellow  chromolipoids  in  chloroplastids  has  been  recognized  by  other 
investigators  who  have  proposed  other  systems  of  classification.  These 
latter  studies  will  be  reviewed  first. 

Tschirch  found  proof  of  the  existence  of  more  than  one  yellow 
chromolipoid  in  his  well-known  series  of  spectroscopic  studies  of 
chlorophyll.  In  his  first  papers  Tschirch  (1884,  1885,  1887)  con- 
sidered the  yellow  constituents  of  the  chloroplastid  to  be  erythrophyll 
(adopting  Bougarel's  terminology)  and  a  group  of  five  xanthophylls, 


38  CAROTINOIDS  AND  RELATED  PIGMENTS 

which  he  called  a,  (3,  Y;  8  and  c  xanthophyll,  respectively,  although  he 
concluded  from  the  fact  that  each  pigment  showed  two  absorption 
bands  showing  no  significant  spectro-analytical  differences  that  they 
were  probably  identical  substances.  Kohl  (1902c)  later  considered 
all  these  xanthophylls  to  be  carotin,  but  a  comparison  of  the  meas- 
urements of  the  spectrum  bands  of  y  and  8  xanthophyll  as  given  by 
Tschirch  (1885)  would  indicate  that  the  former  may  have  been  due 
to  carotin,  but  that  the  latter  was  undoubtedly  one  of  the  xantho- 
phylls as  now  recognized. 

In  his  next  paper  Tschirch  (1896)  regarded  all  the  yellow  chromo- 
lipoids  as  xanthophylls  and  distinguished  between  two,  one  of  which 
was  obtained  in  metallic  glistening  crystals,  which  he  called  xantho- 
carotin.  This  pigment  showed  three  beautiful  absorption  bands,  the 
measurements  of  which  correspond  with  those  now  recognized  for 
xanthophyll.  The  other  xanthophyll  could  not  be  obtained  in  crystal- 
line form  and  its  solutions  were  characterized  by  showing  no  absorp- 
tion bands,  only  end  absorption  of  the  violet  and  ultra-violet. 
Tschirch  used  fresh  grass  as  the  source  of  his  material  for  this  study. 

In  his  most  recent  paper  Tschirch  (1904)  turned  his  attention  to  a 
comparison  of  his  xanthocarotin  and  xanthophyll  with  the  carotin 
from  carrots.  Spectroscopic  absorption  properties  only  were  considered. 
There  has  always  been  a  question  in  the  author's  mind  as  to  which 
group  of  carotinoids  Tschirch's  xanthocarotin  belongs.  Tschirch  him- 
self considered  that  it  might  be  identical  with  the  carotin  from  car- 
rots, inasmuch  as  the  absorption  spectra  of  the  crystalline  pigment 
which  he  isolated  from  carrots  and  that  of  his  xanthocarotin  from 
grass  were  identical.  Kohl  (1902d)  believed  that  Tschirch's  xantho- 
carotin was  carotin  contaminated  with  phytosterin,  and  Tswett 
(1911a)  apparently  also  regarded  the  pigment  as  a  carotin  although 
he  recognized  the  absorption  spectra  of  Tschirch's  carrot  carotin  did 
not  correspond  exactly  with  the  measurements  given  by  other  inves- 
tigators. Willstatter  and  Mieg  (1907)  also  regard  the  xanthocarotin 
as  carotin,  but  with  these  views  the  author  is  not  in  agreement  on  the 
following  grounds.  The  author  believes  that  Tschirch's  carotin  crys- 
tals from  carrots  were  xanthophyll,  not  carotin,  for  he  obtained  them 
merely  by  spontaneous  evaporation  of  an  ether  extract  of  sugar-free 
carrots,  which  would  be  more  likely  to  yield  xanthophyll  crystals  than 
carotin.  Moreover,  the  crystals  had  the  reddish  yellow  color  and  steel 
blue  reflection  described  by  Willstatter  and  Mieg  for  crystals  of  xantho- 
phyll.   In  addition  the  absorption  spectra  of  these  crystals  correspond 


CAROTINOIDS  IN  THE  PHANEROGAMS  39 

exactly  with  those  of  xanthophyll,  not  carotin,  as  the  following  table 
shows : 

Carotin  in  alcohol  Xanthophyll  in  alcohol 

(Willstatter  and  Stoll  (1913a)  (Willstatter  and  Mieg  (1907) 

1—492-476    nil  1—488-471    nfx 

11—459-445     "  11—454-440     " 

III— 430-419     "  III— 4257420     " 

Carrot  carotin  in  alcohol  Xanthocarotin  in  alcohol 

(Tschirch)  (Tschirch) 

1—487-470    fxn  1—485-468    inx 

11—457-439     "  11—455-438     " 

III— 429-417     "  III— 430-418     " 

Much  discussion  has  also  resulted  from  the  statement  made  by 
Tschirch  in  the  paper  under  consideration  that  he  was  able  to  observe 
the  transformation  of  xanthocarotin  into  xanthophyll.  As  a  matter 
of  fact  Tschirch  observed  merely  that  certain  impure  xanthocarotin 
solutions  lost  their  absorption  bands  without  losing  their  color  appre- 
ciably. In  view  of  the  fact  that  the  so-called  xanthophyll  of  Tschirch 
showed  no  absorption  bands  but  merely  end  absorption,  he  concluded 
that  xanthocarotin  readily  changes  over  into  xanthophyll,  a  most 
sweeping  conclusion  from  such  indefinite  evidence.  The  author  has 
observed  many  times  that  impure  solutions  of  carotinoids  lose  their 
spectroscopic  absorption  bands  in  the  earliest  stages  of  decomposition 
with  little  or  no  loss  in  color  of  the  solutions. 

A  somewhat  different  system  of  yellow  chromolipoids  was  proposed 
by  Schunck  (1899,  1901,  1903)  in  his  series  of  papers.  He  depended 
largely  upon  the  spectroscopic  absorption  properties  of  the  pigments 
for  their  differentiation,  as  did  Tschirch,  and  in  his  later  studies  upon 
the  action  of  certain  chemical  agents  upon  the  absorption  bands.  It 
may  be  stated  of  Schunck's  work,  faulty  as  it  was  in  certain  respects, 
particularly  in  his  adoption  of  Sorby's  method  for  separating  the 
various  yellow  coloring  matters  by  carbon  disulfide,  that  he  has  given 
us  some  of  the  most  beautiful  spectro-photographs  of  the  carotinoids 
that  exist  in  the  literature.  Schunck  accepted  from  the  outset  that 
more  than  one  yellow  chromolipoid  was  present  in  the  chloroplastids. 
Inasmuch,  however,  as  he  modified  his  views  somewhat  regarding  the 
number  and  nomenclature  of  these  pigments  during  the  course  of  his 
studies  his  final  views  only  will  be  discussed. 

Schunck  proposed  to  call  all  the  yellow  pigments  accompanying 
chlorophyll  xanthophylls,  the  chief  member  of  the  group  being  chryso- 
phyll,  thus  adopting  Hartsen's  terminology  for  carotin  in  spite  of  the 
fact  that  Schunck  not  only  referred  to  Arnaud's  work  but  confirmed 


40  CAROTINOIDS  AND  RELATED  PIGMENTS 

it  from  a  spectroscopic  standpoint.  Besides  chrysophyll,  the  only 
member  of  the  "xanthophylls"  which  he  was  able  to  obtain  in  crystal- 
line form,  Schunck  separated  two  other  xanthophylls  from  green  leaves 
by  shaking  the  alcoholic  chlorophyll-free  ^  solution  with  successive 
equal  portions  of  carbon  disulfide,  each  volume  of  carbon  disulfide 
being  equal  to  about  one-half  the  volume  of  the  crude  solution  experi- 
mented upon.  This  was  continued  until  no  more  color  was  extracted, 
three  or  four  extractions  being  sufficient  as  a  rule  to  accomplish  this 
result. 

With  the  exception  of  the  first  carbon  disulfide  extract,  which  con- 
tained the  crystallizable  chrysophyll  as  well  as  one  of  the  xantho- 
phylls, Schunck  erroneously  believed  that  the  various  carbon  disul- 
fide fractions  represented  more  or  less  pure  solutions  of  individual 
xanthophylls  with  varying  degrees  of  relative  solubility  in  alcohol  and 
carbon  disulfide. 

The  various  carbon  disulfide  fractions  were  now  allowed  to  evapo- 
rate spontaneously,  the  residue  was  taken  up  again  in  alcohol  and 
the  spectroscopic  absorption  bands  photographed.  The  effect  on  these 
bands  of  adding  concentrated  HCl,  HNO3,  H2SO4,  H2O2  and  nascent 
hydrogen  was  studied,  as  well  as  the  effect  of  these  reagents  on  the 
color  of  the  alcoholic  solution.  Certain  marked  differences  were 
observed  with  the  various  fractions. 

The  first  fraction  from  green  leaves  contained,  besides  chrysophyll, 
a  pigment  which  Schunck  called  L.  xanthophyll,  whose  spectroscopic 
absorption  bands  differed  from  those  of  chrysophyll  by  being  shifted 
only  slightly  towards  the  ultra-violet  and  whose  solution,  like  chryso- 
phyll, changed  to  a  green  tint  before  fading  on  addition  of  HCl  or 
HNO3,  the  absorption  bands  disappearing. 

The  subsequent  carbon  disulfide  extracts  contained  a  second  xan- 
thophyll, called  B.  xanthophyll,  which  differed  from  the  first  in  two 
respects,  (1)  the  absorption  bands  (Schunck  observed  three  distinct 
bands  for  all  his  "xanthophylls")  were  shifted  slightly  more  towards 
the  ultra-violet,  (2)  the  effect  of  acids  on  the  alcoholic  solution  was 
to  produce  a  brilliant  green  color  which  gradually  changed  to  a  beau- 
tiful peacock  blue,  then  purple,  and  gradually  bleached  entirely. 
Especially  striking  was  the  observation  that  the  addition  of  ammonia 

'  This  was  obtained  by  one  of  two  methods,  either  by  adsorbing  the  chlorophyll  on 
animal  charcoal,  which  does  not  remove  the  "xanthophylls,"  according  to  Schunck,  or 
by  saponifying  the  alcoholic  leaf  extract  and  extracting  the  soap  with  ether,  the  latter 
taking  out  the  yellow  pigments.  After  evaporation  of  the  ether  the  pigments  were  taken 
up  in  alcohol  for  the  "xanthophyll"  separations. 


CAROTINOIDS  IN  THE  PHANEROGAMS  41 

to  the  blue  solution  restored  the  original  yellow  color  of  the  solution, 
although  less  intense,  the  blue  color  reappearing  on  acidifying  again. 
Sorby  (1873)  mentioned  this  reaction  for  his  ''yellow  xanthophyll." 
The  author  ^  has  observed  that  the  change  from  yellow  to  blue  and 
vice  versa  can  be  repeated  apparently  indefinitely  with  one  of  the 
xanthophylls  obtained  from  plants  by  Tswett's  chromotographic 
method. 

In  his  last  paper  Schunck  found  evidence  of  the  existence  in  flowers 
of  still  another  xanthophyll,  called  Y.  xanthophyll,  with  properties 
similar  to  B.  xanthophyll,  except  that  it  was  much  less  readily 
extracted  from  alcohol  by  carbon  disulfide  and  was  accordingly  found 
in  the  alcohol  after  the  carbon  disulfide  extractions.  Schunck  found 
no  evidence  of  the  existence  of  Y.  xanthophyll  in  his  leaf  extracts. 

Kohl  (1902e)  attempted  to  harmonize  the  views  of  Tschirch  and 
Schunck  as  well  as  his  own  belief  that  carotin  is  the  principal  pigment 
in  the  chloroplastids.  He  recognized  the  difference  between  carotin 
and  the  xanthophyll  proper  of  Schunck,  but  apparently  did  not  recog- 
nize the  existence  of  several  of  these  xanthophylls,  as  proposed  by 
Schunck.  Kohl  recognized  also  the  existence  of  the  xanthophyll  of 
Tschirch,  which  showed  no  absorption  bands,  and  believed,  like 
Schunck,  who  proposed  no  name  for  the  pigment,  that  it  could  be 
extracted  from  the  chloroplastids  by  hot  water,  as  well  as  by  alcohol. 
Kohl,  therefore,  proposed  to  call  Schunck's  xanthophyll  a  xanthophyll 
and  the  xanthophyll  of  Tschirch  ^  xanthophyll,  and  expressed  the 
belief  that  carotin  and  these  two  xanthophylls  comprised  the  yellow 
pigments  in  the  chloroplastids. 

We  will  now  return  to  a  consideration  of  the  investigations  leading 
up  to  the  classification  of  the  carotinoids  which  prevails  at  the  pres- 
ent time.  Following  Borodin,  Monteverde  (1893)  found  that  the 
yellow  pigments  accompanying  chlorophyll  can  be  divided  into  two 
groups  according  to  their  relative  solubility  in  alcohol  and  petroleum 
ether,  and  he  was  the  first  to  show  that  this  fact  offers  a  very  simple 
means  of  separating  the  pigments  from  each  other.  Using  the  pro- 
cedure of  Fremy  and  Timiriazeff,  Monteverde  precipitated  the  chloro- 
phyll from  an  alcoholic  leaf  extract  with  an  excess  of  Ba(0H)2,  which 
carries  down  with  it  the  carotinoids  also,  and  extracted  the  yellow 
pigments  from  the  precipitate  with  alcohol.  Petroleum  ether  and  a 
few  drops  of  water  were  added  to  this  yellow  solution,  and  the  mix- 
ture shaken.     The  liquids  soon  separated  into  two  layers,  each  con- 

"  Unpublished  observation. 


42  (JAROflNOIDS  AND  RELATED  PIGMENTS 

taining  a  yellow  pigment  with  distinguishing  characteristics.    Monte- 
verde  found  the  pigment  in  the  upper  petroleum  ether  layer  to  be 
spectroscopically  as  well  as  in  other  respects  identical  with  carotin 
and   accordingly   called  it  carotin.     The   pigment  remaining   in  the 
alcohol  layer,  on  the  other  hand,  was  found  to  be  different  in  many 
respects  and  was  called  xanthophyll,  following  Gregor  Kraus'  termi- 
nology.    Monteverde  regarded  it  as  not  unlikely  that  this  "xantho- 
phyll" itself  consisted  of  two  yellow  pigments.    In  order  to  separate 
completely   the    carotin    and   xanthophyll   the   petroleum   ether    and 
alcohol  layers  after  separation  were  shaken  with  fresh  quantities  of 
alcohol  and  petroleum  ether,  respectively.     On  spontaneous  evapora- 
tion of  the  alcoholic  xanthophyll  solutions  Monteverde  obtained  crys- 
tals which  corresponded  exactly  in  form  with  the  "strohgelben  Krys- 
tallen"  described  by  Borodin.    There  is  some  doubt,  however,  whether 
the  pale  yellow  crystals  observed  by  Monteverde,  and  the  similar  ones 
observed  by  Borodin,  were  actually  xanthophyll.    Reinke  (1885)  sev- 
eral years  previously  obtained  yellow  platelets  on  evaporation  of  alco- 
holic solutions  of  the  yellow  chloroplastid  pigments  and  found  them 
to  be  merely  phytosterol  or  a  mixture  of  sterols  colored  with  pigment. 
It  is  likely  that  Monteverde  was  misled  by  the  same  phenomenon,  as 
the  great  solubility  of  xanthophyll  in  alcohol  undoubtedly  prevents 
the  formation  of  crystals  when  one  is  dealing  with  the  very  small 
quantities  of  pigment  present  in  Monteverde's  solutions.    Monteverde, 
however,  described  very  clearly  the  difference  between  the  absorption 
spectra  of  carotin  and  xanthophyll,  as  did  Schunck,  some  years  later, 
between  chrysophyll  (carotin)  and  the  L.  B.  and  Y.  xanthophylls  which 
he  separated.    Monteverde  also  described  the  green  coloration,  chang- 
ing to  a  blue  on  addition  of  concentrated  HCl  to  the  alcoholic  xan- 
thophyll solution,  a  reaction  which  also  characterized  the  B.  and  Y. 
xanthophylls  of  Schunck,  as  mentioned  in  an  earlier  paragraph. 

Tswett  was  very  quick  to  recognize  the  importance  of  Monteverde's 
work  and  the  significance  of  the  Kraus  method  of  separation  in  indi- 
cating the  existence  of  alcohol-soluble  xanthophylls  in  contrast  with 
benzine-soluble  carotin.  This  investigator's  keen  appreciation  of  the 
significant  properties  of  carotin  and  xanthophylls  is  what  makes  pos- 
sible today  the  extension  of  our  knowledge  of  the  distribution  of 
these  pigments  in  all  forms  of  plant  and  animal  matter.  Tswett's 
important  observations  are  accessible  to  us  in  a  series  of  papers 
(1906a,  b,  c,  1911a)  from  1906  to  1911.  The  last  paper  is  more  of 
the  nature  of  a  summary  but  by  reason  of  its  clear-cut  statements  it 


CAROTINOIDS  IN  THE  PHANEROGAMS  43 

may  well  serve  today  as  our  best  laboratory  outline  for  working  with 
the  class  of  pigments  with  which  this  monograph  deals.  It  was  in  this 
paper  that  Tswett  proposed  the  nomenclature  for  the  carotinoids  which 
has  been  adopted  in  this  monograph. 

Tswett's  most  important  contribution  to  the  subject,  from  an  inves- 
tigational standpoint,  was  on  certain  physico-chemical  properties  of 
the  pigments.  He  showed  (1906b)  that  the  various  colored  constitu- 
ents of  the  chloroplastids,  when  carefully  obtained  in  certain  solvents 
by  methods  which  avoid  the  action  of  plant  acids,  exhibit  very  char- 
acteristic adsorption  coefficients  towards  finely  divided  materials,  such 
as  CaCOa,  inulin  and  sucrose,  as  well  as  many  other  inert  materials 
which  are  insoluble  in  the  solvent  employed  and  which  can  be  obtained 
in  a  finely  divided  state.  This  exceedingly  interesting  phenomenon 
is  no  doubt  due  to  the  fact  that  the  various  green  and  yellow  chromo- 
lipoid  constituents  of  the  chloroplastids  exist  in  organic  solvents  in 
colloidal  aggregates  of  various  sizes,  the  larger  colloidal  particles 
being  the  more  strongly  adsorbed,  and  some,  like  carotin,  which  is  not 
adsorbed  at  all,  existing  in  true  solution.  Tswett  found  petroleum 
ether,  the  carotin  solvent,  to  serve  best  for  the  study  of  these  prop- 
erties, although  carbon  disulfide  was  also'  very  useful  because  of  the 
brilliant  color  which  all  the  chloroplastid  pigments  show  in  this  sol- 
vent, and  also  because  the  xanthophylls  are  especially  well  differen- 
tiated in  this  solvent.  This  latter  fact  is  no  doubt  closely  related  to 
Schunck's  (1903)  observations  regarding  the  relative  solubility  of 
xanthophylls  in  carbon  disulfide  by  which  he  believed  he  was  able  to 
separate  them  from  one  another  by  a  shaking-out  method.  Schunck's 
observations  were  near  the  truth  but  can  not  be  compared  in  accuracy 
with  the  method  of  separation  which  Tswett  was  able  to  develop  from 
the  colloidal  properties  of  xanthophylls. 

Tswett  hit  upon  a  very  ingenious  method  indeed  of  applying  the 
results  of  his  study.  He  filtered  the  moisture-free  petroleum  ether 
solution  of  the  mixed  chloroplastid  pigments  (or  carbon  disulfide  solu- 
tion) through  a  column  of  perfectly  dry  CaCOg,  packed  as  tightly  and 
evenly  as  possible  in  a  glass  tube,  and  found  that  the  various  pig- 
ments differentiated  themselves  according  to  their  adsorption  affinity 
(colloidal  aggregation)  for  the  CaCOg.  The  resulting  chromatogram 
(as  Tswett  proposed  to  call  it)  presented  a  most  surprising  picture  of 
the  chloroplastid  pigments,  which  is  strikingly  similar  in  effect,  if  not 
in  principle,  to  the  well-known  Liesegang  phenomena. 

By  applying  this  chromatographic  method  of  analysis  to  petroleum 


44  CAROTINOIDS  AND  RELATED  PIGMENTS 

ether  and  carbon  disulfide  solutions  of  the  chloroplastid  pigments  from 
plantain  {Plantago)  and  dead  nettle  {Lamium  album)  leaves  Tswett 
has  shown  that  at  least  three  and  possibly  four  xanthophylls  accom- 
pany carotin.  He  has  provisionally  designated  these  a,  a,  a',  and  p 
xanthophylls,  respectively.  Tswett  has  characterized  these  pigments 
further,  as  follows: 

Xanthophyll  a.  This  pigment  is  least  adsorbed  by  the  CaCOs  and 
is  closest  to  carotin  in  this  respect,  which  is  not  adsorbed  at  all.  Its 
adsorption  zone  is  the  lowest  in  the  column  of  the  xanthophyll  zones 
and  has  an  orange-yellow  color  when  carbon  disulfide  is  the  solvent. 
It  is  hypophasic  in  the  Kraus  separation,  i.e.,  remains  in  the  alcohol 
layer.  It  shows  three  well  marked  absorption  bands,  the  first  two  of 
which,  in  alcohol  or  petroleum  ether  solution,  lie  at  485-470ix|i,  and 
455-440^x[x.  Its  alcoholic  solutions  are  merely  bleached  on  addition  of 
con.  HCl. 

Xanthophylls  a'  and  a'.  These  pigments  lie  very  close  together 
in  the  column  but  above  the  zone  of  xanthophyll  a.  In  CSg  their 
zones  are  yellow.  They  are  similar  in  properties  to  xanthophyll  a, 
i.e.,  in  the  Kraus  separation  and  spectroscopically,  but  their  absorp- 
tion bands  are  shifted  slightly  towards  the  violet.  The  effect  of  HCl 
on  the  alcoholic  solutions  is  not  mentioned  but  the  author  (1914g) 
has  found  that  for  xanthophyll  a,  at  least,  no  color  reaction  is 
produced. 

Xanthophyll  (3.  This  pigment  shows  the  greatest  adsorption  affinity 
for  CaCOs  (exists  in  the  largest  colloidal  aggregates)  and  comprises 
the  highest  yellow  zone  in  the  column.  This  pigment  is  hypophasic 
in  the  Kraus  separation  like  the  other  xanthophylls,  but  may  be  dif- 
ferentiated from  them  by  the  fact  that  its  alcoholic  solution  gives  a 
blue  color  on  addition  of  con.  HCl,  and  also  by  the  fact  that  its 
absorption  bands  are  shifted  perceptibly  towards  the  violet  from  those 
of  xanthophylls  a,  a,  and  a^,  the  first  two  bands  lying  at  475-462^^ 
and  445-430|i^A,  when  in  alcoholic  solution.  The  xanthophyll  (3  of 
Tswett  appears  to  be  identical  with  the  ''yellow  xanthophyll"  of  Sorby 
and  the  Y.  xanthophyll  of  C.  A.  Schunck,  but  bears  no  relation  what- 
ever to  the  xanthophyll  p  of  Kohl.  According  to  Tswett  (1908b)  the 
latter  is  not  a  xanthophyll  at  all,  in  fact  does  not  exist  in  the  plant 
but  is  merely  a  post-mortem  decomposition  product  derived  from 
colorless  chromogens  whose  alkali  salts  are  yellow  and  which  assume 
a  dark  color  on  oxidation. 

The  relative  solubility  properties  of  carotin  and  xanthophylls  as 


CAROTINOIDS  IN  THE  PHANEROGAMS  45 

exhibited  in  the  Kraus  separation  indicated  to  Tswett  (1906a)  a  fun- 
damental chemical  difference  between  the  two  groups  of  carotinoids. 
The  proof  of  this  theory  as  well  as  the  nature  of  the  difference  was 
soon  brought  to  light  by  Willstatter  and  Mieg  (1907)  when  they  iso- 
lated the  first  crystalline  xanthophyll  and  submitted  it  to  analysis. 
Working  on  the  same  elaborate  scale,  which  has  characterized  all  the 
researches  on  carotinoids  in  Willstatter's  laboratory,  a  crystalline 
xanthophyll  was  isolated  from  100  kilos  of  dried  nettle  (Urtica)  leaves. 
The  average  of  five  ultimate  analyses  of  crystals  prepared  both  by 
recrystallization  from  methyl  alcohol  and  from  chloroform  (by  addi- 
tion of  petroleum  ether)  showed  84.22  per  cent  carbon  and  9.92  per 
cent  hydrogen,  which  corresponds  very  closely  with  the  theoretical 
values  of  84.44  per  cent  carbon  and  9.93  per  cent  hydrogen  for  the 
formula  C4oH5e02.  This  was  confirmed  fairly  well  by  a  molecular 
weight  determination  (found  512,  theory  564),  and  better  by  an 
analysis  of  the  iodine  content  of  the  theoretically  simplest  iodine  addi- 
tion product,  C4oH5,;02l2  (found  31.68  per  cent,  theory  30.86  per  cent). 

The  chemical  properties  of  the  crystalline  xanthophyll  isolated  by 
Willstatter  and  Mieg  will  be  considered  in  detail  elsewhere.  Several 
points,  however,  may  profitably  be  considered  at  this  point.  The 
crystalline  product  showed  the  greatest  solubility  difference  from  caro- 
tin in  alcohol  and  low  boiling  petroleum  ether,  being  practically  in- 
soluble in  the  latter,  but  readily  soluble  in  the  former,  which  is  just 
the  reverse  of  carotin  in  these  solvents.  The  Kraus  method  of  separa- 
tion of  the  pigments  was  further  confirmed  by  Willstatter  and  Mieg 
by  applying  the  test  in  several  ways  to  solutions  of  the  purified  pig- 
ments. The  difference  between  the  position  of  the  absorption  bands 
of  carotin  and  the  xanthophylls,  first  pointed  out  by  Monteverde,  was 
confirmed,  the  first  two  bands  as  measured  by  Willstatter  and  Mieg 
lying  at  480-470!itx  and  453-437^^. 

Willstatter  and  Mieg  expressed  their  belief  in  the  existence  of  a 
group  of  xanthophylls  in  the  paper  under  consideration  although  they 
were  apparently  not  familiar  with  Tswett's  demonstration  of  this  fact 
a  year  before  their  paper  appeared.  The  question  naturally  arises  as 
to  which  xanthophyll  was  obtained  in  crystalline  form  by  these  in- 
vestigators. 

Tswett  (1910a)  has  expressed  the  opinion  that  the  xanthophyll 
crystallized  by  Willstatter  and  Mieg  was  a  mixture  of  two  or  three 
xanthophylls  in  which  xanthophyll  d  predominated,  a  possibility  which 
was  later  acknowledged  by  Willstatter  and  Stoll  (1913b).     The  evi- 


46  CAROTINOIDS  AND  RELATED  PIGMENTS 

.  dence  available  on  this  question  indicates,  however,  that  xanthophyll 
(3  may  have  formed  a  considerable  proportion  of  the  crystalline  prep- 
aration. Willstatter  and  Mieg  mention  the  fact  that  their  prepara- 
tion dissolved  in  strongly  alcoholic  HCl  with  a  blue  color,  a  reaction 
which  is  apparently  characteristic  of  xanthophyll  p  only.  In  the  Sorby 
and  C.  A.  Schunck  separation,  however,  the  pure  pigment  differen- 
tiated itself  almost  equally  between  the  alcohol  and  carbon  disulfide 
layers,  a  reaction  which  obviously  characterizes  the  a  group  of  xan- 
thophylls  because  of  their  lesser  adsorption  from  this  solvent  by 
CaCOg.  Still  further  evidence  of  a  mixture  of  xanthophylls  in  the 
Willstatter  and  Mieg  preparation  is  the  fact  that  its  spectroscopic 
absorption  bands  apparently  lie  in  an  intermediary  position  between 
the  bands  of  xanthophylls  a  and  (3  as  recorded  by  Tswett. 

The  isolation  of  the  various  members  of  the  xanthophyll  group  in 
crystalline  form  seems  greatly  to  be  desired  in  order  that  the  dif- 
ferences existing  between  the  individual  members  of  this  class  of 
carotinoids  may  be  determined.  The  relative  adsorption  properties 
of  these  pigments  offers  the  most  promising  method  for  accomplishing 
this  result  but  the  experimental  work  would  have  to  be  conducted  on 
a  very  generous  scale.  The  xanthophylls  are  unquestionably  either 
isomorphic  or  isomeric  forms  of  the  same  empirical  composition, 
C40H56O2,  as  Willstatter  and  Stoll  (1913)  have  pointed  out.  The 
author  believes  that  Willstatter  and  Escher  (1912)  have  already  iso- 
lated pure  xanthophyll  a  in  the  form  of  their  so-called  lutein  from' 
egg  yolk,  as  will  be  discussed  more  fully  in  a  later  chapter. 

It  is  not  likely  that  more  than  four  xanthophylls  characterize  the 
chioroplastid  for  the  author  (1914g)  has  found  only  four  on  applying 
the  chromatographic  method  to  extracts  from  an  entirely  different 
plant  than  Tswett  used,  namely,  the  leaves  of  alfalfa  (Medicago 
sativa).  The  possibility  of  other  xanthophylls  being  present  in  non- 
chlorophyllous  organs  is  indicated,  however,  by  a  chromatographic 
analysis  which  the  author  (1914g)  carried  out  on  the  xanthophyll 
fraction  (obtained  by  the  Kraus  separation)  of  the  pigments  of  the 
carrot  root,  in  which  no  less  than  eight  distinct  yellow  or  orange 
zones  characterized  the  chromatogram.  The  possibility  remains  to 
be  investigated,  however,  whether  this  result  was  influenced  in  any 
way  by  the  method  of  preparation  of  the  materia"!  or  other  experi- 
mental steps  in  the  procedure  employed.  The  author  regards  the 
adsorption  phenomenon  of  the  carotinoids  as  colloidal  so  that  it  may 
not  be  possible  to  secure  these  pigments  in  every  case  in  the  same 


CAROTINOIDS  IN  THE  PHANEROGAMS  47 

degree  of  colloidal  aggregation.  Willstatter  and  Mieg  found  that 
their  crystalline  xanthophyll  readily  entered  into  combination  with 
solvents  forming  molecules  of  solvent  of  crystallization,  which  is 
unquestionably  a  colloidal  combination  and  might  easily  influence 
greatly  the  adsorption  properties  of  the  pigments.  The  whole  adsorp- 
tion phenomenon  deserves  a  further  study  using  pure  preparations  of 
the  individual  pigments. 

The  xanthophylls  are  usually  regarded  as  pigments  in  which  yellow 
is  the  predominating  color.  Red  colored  xanthophylls  also  exist, 
however.  Monteverde  (1893)  first  called  attention  to  a  red  pigment 
in  the  reddish-brown  leaves  of  the  young  floating  pond  weed  {Potamo- 
geton  natans) ,  an  aquatic  herb  widely  distributed  in  Russia,  which 
showed  the  xanthophyll  properties  in  the  Kraus  separation.  This  pig- 
ment has  since  been  called  rhodoxanthin  by  Monteverde  and  Lubi- 
menko  (1913b),  who  obtained  it  in  crystalline  form.  The  pigment 
appears  to  be  isomeric  with  the  xanthophyll  of  the  chloroplastids,  as 
lycopin  is  isomeric  with  carotin.  It  differs  from  the  usual  yellow 
xanthophyll  by  dissolving  in  formic  acid  with  a  yellow  color,  yellow 
xanthophyll  dissolving  in  this  solvent  with  a  green  color,  according 
to  Monteverde  and  Lubimenko.  Rhodoxanthin  also  shows  spectro- 
scopic absorption  bands  with  characteristic  position,  especially  in  car- 
bon bisulfide.  A  comparison  of  the  xanthophyll  and  rhodoxanthin 
bands  in  this  solvent,-  as  given  by  Willstatter  and  Stoll  (1913)  and  by 
Monteverde  and  Lubimenko,  respectively,  is  shown  in  the  following: 

Xanthophyll  (W.  and  S.)  Rhodoxanthin  (M.  and  L.) 
Band  I        516-501    m-M-  575-553   m-!^ 

Band  II      483-467     "  535-515     " 

Band  III    447-441     "  500-480     " 

The  general  solubility  properties  of  rhodoxanthin  appear  to  follow 
those  of  xanthophyll  very  closely. 

The  relation  between  the  empirical  constitution  of  carotin  and  the 
xanthophylls  is  such  that  the  latter  may  be  expressed  very  simply  as 
carotin  dioxides.  The  character  of  the  oxygen  combination,  however, 
is  not  clear,  for  according  to  the  statement  of  Willstatter  and  Mieg 
their  crystalline  xanthophyll  did  not  show  the  presence  of  either 
hydroxy,  carboxyl  or  carbonyl  groups.  The  xanthophylls,  therefore, 
cannot  be  simple  oxidation  products  of  carotin.  But  these  statements 
regarding  the  character  of  the  oxygen  in  the  xanthophyll  molecule 
possibly  should  be  confirmed,  for  notwithstanding  the  fact  that  it  has 
not  yet  been  found  possible  to  transform  carotin  into  xanthophyll  in 


48  CAROTINOIDS  AND  RELATED  PIGMENTS 

the  laboratory,  the  constant  presence  of  these  pigments  in  the  chloro- 
plastid  is  very  difficult  to  explain  unless  the  constitution  of  one  type 
of  pigment  bears  a  simple  relation  to  that  of  the  other.  The  various 
theories  which  have  been,  offered  regarding  the  possible  functions  of 
the  carotinoids  in  the  chloroplastid  also  fall  down  unless  the  caro- 
tinoids  are  closely  relationed  chemically.  Ewart  (1915),  to  be  sure, 
has  recently  claimed  to  have  succeeded  in  reducing  xanthophyll  to 
carotin  in  the  laboratory.  The  evidence  for  this  is  very  unconvincing, 
especially  in  view  of  the  fact  that  Ewart  on  subsequent  study  (1918) 
failed  to  substantiate  any  of  the  other  products  which  he  first  claimed 
to  have  produced  from  xanthophyll  on  photo-oxidation.  The  reduc- 
tion experiment  of  xanthophyll  to  carotin  unfortunately  was  not 
repeated  in  the  second  study. 

Carotinoids  in  Etiolated  Leaves 

The  yellow  chromolipoids  which  develop  without  chlorophyll  in  the 
leucoplastids,  when  plants  are  grown  in  the  dark,  would  seem  to  be 
closely  related  to,  if  not  completely  identical  with  those  found  in  the 
chloroplastids,  at  least  qualitatively,  inasmuch  as  etiolated  plants  form 
chlorophyll  very  rapidly  in  the  light  without  loss  of  yellow  constitu- 
ents in  the  resulting  chloroplastids.  No  studies  have  been  made  of  the 
pigments  of  etiolated  leaves,  however,  since  our  newer  chemical  con- 
ceptions of  the  plant  carotinoids  have  arisen  so  that  it  is  necessary 
to  depend  upon  older  investigations  for  our  experimental  knowledge 
of  these  coloring  matters.  It  is  possible  to  state  with  certainty  that 
carotin  is  present  in  the  etiolated  plant,  but  the  evidence  is  insuffi- 
cient to  substantiate  the  belief  of  Tammes  (1900)  and  Kohl  (1902f) 
that  it  is  probably  the  only  yellow  chromolipoid  present  inasmuch  as 
it  is  now  known  that  the  methods  which  these  investigators  employed 
are  not  specific  for  carotin. 

Joannes  Rajus  (1693)  appears  to  have  first  recorded  the  observation 
that  plants  which  grow  in  the  dark  do  not  turn  green  but  have  a 
yellow  color.  Bonnet  (1754)  named  such  plants  "plantes  etiolees." 
The  question  whether  the  yellow  pigment  or  some  colorless  substance 
was  the  forerunner  of  the  green  pigment  which  developed  so  rapidly 
when  etiolated  plants  are  exposed  to  the  light  occupied  the  attention 
of  many  investigators.  Interest  in  this  question  was  stimulated  by  the 
discovery  of  Phipson  (1858)  that  etiolated  leaves  rapidly  assume  an 
emerald  green  color  when  immersed  in  con.  H2SO4.    Phipson  followed 


CAROTINOIDS  IN  THE  PHANEROGAMS  49 

the  terminology  of  Berzelius  for  yellow  autumn  pigments  and  called 
the  etiolated  pigment  xanthophyll.^  Fremy  (1860)  naturally  regarded 
the  yellow  pigment  of  young  sprouts  and  etiolated  leaves  as  identical 
with  his  phylloxanthine  and  the  bluish-green  color  which  develops  on 
treatment  with  acid  (he  also  found  the  fumes  of  HCl  and  HNO3  very 
effective)  as  identical  with  his  phyllocyanine.  Sorby  (1871b)  recog- 
nized the  relation  of  the  yellow  pigment  of  etiolated  leaves  to  other 
yellow  plant  pigments  and  regarded  the  color  as  due  to  a  preponder- 
ance of  his  so-called  "xanthophyll"  group,  characterized  by  their 
solubility  in  carbon  disulfide  and  two  more  or  less  distinct  spectro- 
scopic absorption  bands  in  the  blue  part  of  the  spectrum.  Gregor 
Kraus  (1872b)  compared  the  spectroscopic  properties  of  the  alcoholic 
extract  of  etiolated  leaves  with  his  xanthophyll  pigment  which  re- 
mained in  the  alcohol  on  shaking  leaf  extracts  with  benzene.  The 
results  led  him  to  believe  that  the  pigments  were  probably  identical, 
and  he  proposed  a  genetic  relation  of  the  etiolated  pigment  to  the 
green  pigment  of  plants. 

Pringsheim  (1874),  however,  also  using  a  spectroscopic  examination 
of  the  alcoholic  extracts  of  etiolated  leaves  as  the  basis  of  his  con- 
clusions, found  characteristic  absorption  bands  in  the  red  end  of  the 
spectrum  in  addition  to  bands  in  the  blue  which  characterized  Kraus' 
xanthophyll.  Inasmuch  as  the  same  result  was  obtained  for  each  of 
10  different  etiolated  plants  which  he  examined,  Pringsheim  concluded 
that  a  special  pigment  was  present  which  caused  the  bands  in  the  red 
as  well  as  the  bands  in  the  blue.  He  called  this  pigment  etiolin. 
Pringsheim's  results  have  been  frequently  substantiated,  and  while 
some  subsequent  investigators  (Wiesner  (1877b),  Elfving  (1882), 
Tschirch  (1884))  have  agreed  with  his  conclusion  regarding  etiolin  as 
a  distinct  pigment,  a  majority  (Timiriazeff  (1875),  Hansen  (1884b), 
Immendorff  (1889),  Monteverde  (1894),  Kohl  (1902f),  Greilach 
(1904)  who  have  studied  this  phase  of  the  etiolin  question  have  pre- 
sented convincing  evidence  that  the  spectroscopic  absorption  bands  in 
the  red  which  Pringsheim  observed  in  his  etiolated  leaf  extracts  are 

'According  to  Czapek  (Biochemie  der  Pflanzen,  2nd  Ed.,  vol.  I,  p.  579,  Jena,  1913), 
Julius  Sachs  (1859  a,  b)  and  Jos.  Boehm  (1859)  called  the  etiolated  pigment  leuko- 
phyll  and  chlorogon,  respectively.  The  statement  is  incorrect.  The  leukophyll  of 
Sachs  was  a  colorless  chromogen  in  the  seeds  and  also  in  the  etiolated  plants  which 
gave  rise  to  the  green  chlorophyll  in  the  sunlight  or  on  treatment  with  acids  (com- 
pare Phipson  [1858]),  while  the  chlorophor  (not  chlorogon  as  Czapek  has  it)  of  Boehm 
was  the  same  colorless  chromogen.  Boehm  differed  from  Sachs  in  regarding  the  green 
acid  derivative  of  the  colorless  chromogen  as  an  artificial  pigment  and  the  green  sun- 
light derivative  as  the  true  chlorophyll.  Both  investigators  recognized  the  existence  of 
the  yellow  etiolated  leaf  pigment  as  well  as  the  colorless  chromogen. 


50  CAROTINOIDS  AND  RELATED  PIGMENTS 

either  chlorophyll  or  a  closely  related  forerunner  of  one  of  the  chloro- 
phyllins.  Timiriazeff  (1875)  believed  the  absorption  spectra  of  alco- 
holic etiolated  leaf  extracts  to  be  due  to  a  small  amount  of  chlorophyl- 
lin  admixed  with  Kraus'  xanthophyll.  Hansen  (1884b)  regarded  the 
bands  in  the  red  as  due  to  chlorophyll.  Monteverde  (1894)  regarded 
the  substance  giving  the  bands  in  the  red  as  a  forerunner  of  one  of 
the  chlorophyllins  and  called  it  protochlorophyll,  a  view  which  seems 
to  have  been  substantiated  by  the  work  of  Greilach  (1904).  The 
latter  proposes  to  reserve  the  name  etiolin  for  this  green  pigment  with 
properties  like  chlorophyll  which  exists  in  etiolated  leaves  in  very 
small  amounts,  and  to  use  the  term  in  the  same  sense  as  Monteverde 
used  the  word  protochlorophyll.  According  to  Greilach  etiolin  (proto- 
chlorophyll) is  not  a  .constant  constituent  of  the  etiolated  leaf  but 
appears  and  then  disappears  during  the  germination  of  the  seed  in 
the  dark. 

Arnaud  (1889),  following  his  earlier  (1885)  demonstration  regard- 
ing the  identity  of  the  yellow  leaf  pigment  isolated  by  him  with  the 
carotin  from  carrots,  regarded  the  yellow  color  of  etiolated  leaves  as 
due  to  the  same  pigment.  No  chemical  proof  was  offered  of  this  but 
he  determined  the  quantity  of  carotin  in  the  etiolated  leaves  of  the 
kidney  bean  {Phaseolus  vulgaris),  using  a  colorimetric  method  which 
will  be  reviewed  in  a  later  chapter.  Inasmuch  as  Arnaud's  method 
of  analysis  would  preclude  all  but  traces  of  xanthophylls  his  result 
may  be  regarded  as  the  first  proof  of  the  presence  of  carotin  in  etio- 
lated leaves.  This  was  confirmed  completely  by  Immendorff  (1889) 
the  same  year.  He  saponified  the  alcoholic  extracts  from  etiolated 
leaves,  extracted  the  carotinoids  from  the  soap  with  ether  and  obtained 
crystals  of  carotin  from  the  golden  yellow  extract.  He  did  not  suc- 
ceed in  obtaining  crystals  from  etiolated  leaves  which  had  developed 
only  a  pale  yellow  color,  but  only  from  those  having  a  more  orange 
color,  but  this  cannot  be  interpreted  as  indicating  another  pigment  in 
the  less  pigmented  leaves,  as  Immendorff  believed,  but  must  be  re- 
garded as  due  solely  to  differenoes  in  concentration  of  pigment,  as  Kohl 
(1902f)  has  pointed  out. 

Following  Immendorff,  Molisch  (1896),  Tammes  (1900)  and  Kohl 
(1902f)  have  independently  substantiated  the  presence  of  carotinoids 
in  etiolated  leaves  from  various  plants  using  microscopic  crystalliza- 
tion methods  on  the  fresh  tissues.  Inasmuch  as  our  information  re- 
garding the  yellow  chromolipoids  in  the  etiolated  leaf  depends  at  the 
present  time  on  the  observations  of  these  authors  and  the  microchemi- 


CAROTINOIDS  IN  THE  PHANEROGAMS  51 

cal  methods  which  they  used,  it  will  be  necessary  to  state  briefly  the 
character  and  significance  of  the  methods,  reserving  a  fuller  descrip- 
tion for  a  later  chapter. 

Frank  (1884)  first  observed  that  red  crystalline  needles  form  in  the 
plastids  and  between  the  chlorophyll  granules  when  green  leaves  are 
immersed  in  dilute  acids  for  a  time,  and  then,  after  washing  off  the 
acid,  are  allowed  to  remain  in  distilled  water  for  a  still  more  pro- 
tracted period.  Tschirch  (1884),  who  first  examined  the  phenomenon, 
did  not  decide  the  nature  of  the  crystals,  but  Molisch  (1896)  found  the 
crystals  to  be  identical  in  properties,  although  having  a  more  reddish 
color,  with  the  majority  of  crystals  which  he  found  could  be  produced 
by  an  entirely  different  method.  Molisch's  method  is  to  immerse  the 
leaves  in  dilute  (40  per  cent  by  volume)  alcohol  containing  20  per 
cent  KOH,  until  the  chlorophyll  is  completely  extracted.  The  process 
sometimes  requires  several  days.  On  washing  off  the  green  extract 
with  water,  and  immersing  the  washed  leaves  in  distilled  water  for 
several  hours  to  insure  the  complete  removal  of  the  chlorophyll,  it  is 
found  that  crystals  of  various  forms  and  colors  from  yellowish-orange 
to  red  have  appeared  abundantly  in  the  leaf.  Molisch  proved  fairly 
conclusively  the  identity  of  many  of  the  crystals  thus  obtained  with 
the  red-orange  crystals  which  form  in  concentrated  alcoholic  leaf 
extracts,  and  accordingly  decided  to  call  the  crystals  carotin.  Molisch 
was  careful  to  point  out,  however,  that  he  used  the  term  carotin  in  the 
sense  of  a  group  of  closely  related  pigments,  for  he  recognized  that  the 
crystals  formed  by  his  alkali  method  were  not  due  in  all  cases  to  the 
same  pigment.  Tammes  (1900)  and  Kohl  (1902),  however,  who 
greatly  extended  our  knowledge  of  the  presence  of  carotinoids  in  the 
plant  kingdom,  using  the  microchemical  methods  of  Frank  and 
Molisch,  believed  that  only  one  pigment  was  concerned,  namely,  caro- 
tin, and  regarded  the  methods  as  specific  for  this  pigment.  Tswett 
(19ila),  however,  proved  definitely  that  the  crystals  obtained  by 
Molisch's  method  are  a  mixture  of  carotinoids,  and  this  has  been  com- 
pletely confirmed  by  van  Wisselingh  (1915).  Other  microchemical 
methods  for  carotinoids  have  been  worked  out  by  the  two  investi- 
gators just  mentioned,  and  these  will  be.  reviewed  in  a  later  chapter. 
Tswett  has  stated  that  Frank's  acid  method  may  possibly  be  specific 
for  carotin.  This  may  well  be  the  case  in  view  of  the  much  greater 
sensitiveness  of  the  xanthophylls  to  acids,  as  van  Wisselingh  has 
pointed  out,  but  this  investigator  who  has  studied  the  method  closely 
finds  it  to  be  often  laborious,  requiring  sometimes  several  months  for 


52  CAROTINOIDS  AND  RELATED  PIGMENTS 

the  crystals  to  form,  and  that  it  frequently  fails  to  show  the  presence 
of  carotin  in  plant  tissues  in  which  the  pigment  is  known  to  be  pres- 
ent. By  the  use  of  suitable  solvents  van  Wisselingh  has  demonstrated 
very  ingeniously,  however,  that  it  is  possible  to  distinguish  the  xan- 
thophyll  crystals  as  a  group  from  the  carotin  crystals  in  the  mixture 
formed  by  the  Molisch  method  in  various  plant  tissues. 

Returning  now  to  the  investigations  regarding  the  chromolipoids  in 
various  etiolated  plants,  it  may  be  stated  that  Molisch  (1896)  demon- 
strated carotinoids  by  his  alkali  method  in  the  etiolated  leaves  of 
garden  cress  {Lepidium  sativum),  barley  [Hordeum  vulgare) ,  hemp 
{Cannabis  sativa) ,  oats  {Pisum  sativum),  and  of  balsam  {Balsamina 
hortensis,  D.C.)  and  fir  [Abies  excelsa) ,  but  not  from  the  sunflower 
[Helianthus  annuus) ,  in  which  only  orange-red  drops  formed.  Tammes 
(1900),  using  seeds  from  the  same  plants,  save  the  balsam  and  fir, 
substantiated  Molisch's  positive  results  with  the  alkali  method,  and 
in  addition  obtained  the  carotinoid  crystals  in  the  etiolated  di-cotyle- 
dons  of  the  sunflower.  Kohl  (1902f)  denounced  most  emphatically 
the  view  of  Pringsheim  that  a  specific  pigment  is  present  in  etiolated 
leaves,  devoting  an  entire  chapter  of  his  monograph  to  this  question. 
In  addition  to  the  etiolated  plants  examined  by  Molisch  and  Tammes, 
Kohl  (1902f)  was  able  to  form  carotinoid  crystals  in  the  etiolated 
leaves  of  the  turnip  {Sinapis  alba)  and  various  varieties  of  Asphodel. 

It  is  apparent  that  we  have  as  yet  only  indirect  evidence  that 
xanthophylls  are  present  in  the  etiolated  leaf.  C.  A.  Schunck  (1903) 
has  furnished  direct  evidence  of  xanthophylls  in  an  isolated  case, 
namely,  the  etiolated  leaves  of  the  daffodil  (Narcissus  pseudo-nar- 
cissus),  using  the  carbon  disulfide  separation  method  which  has 
already  been  described.  A  mixture  of  xanthophylls  was  found  to  be 
present,  but  Schunck  was  unable  to  obtain  crystals  of  chrysophyll 
(carotin)  although  he  had  no  difficulty  in  obtaining  them  abundantly 
from  alcoholic  extracts  of  the  etiolated  leaves  which  had  been  allowed 
to  turn  green  in  the  sunlight.  Greilach's  (1904)  spectroscopic  obser- 
vations of  the  pigments  in  etiolated  leaves  led  him  to  conclude  that 
yellow  pigments  other  than  carotin  are  also  concerned  in  the  colora- 
tion of  the  leaves.  Ewart  (1918)  states  that  he  has  found  8  to  10 
parts  of  carotin  to  one  of  xanthophyll  in  etiolated  wheat  seedlings. 

Several  investigators  have  studied  the  question  raised  by  the  last 
observation  of  Schunck,  namely,  what  effect  greening  has  on  the  con- 
tent of  carotinoids  in  the  etiolated  leaf.  Wiesner  (1877a)  first  studied 
this  point  and  concluded  that  the  xanthophyll  (carotinoids)  diminished 


CAROTINOIDS  IN  THE  PHANEROGAMS  53 

during  the  greening  of  etiolated  sprouted  oats.  Inasmuch,  however, 
as  he  used  a  colorimetric  comparison  of  the  total  alcoholic  extract  of 
the  etiolated  plant  with  the  alcoholic  xanthophyll  layer  of  the  extract 
from  the  green  plant  following  the  Kraus  separation,  it  is  not  dif- 
ficult to  account  for  his  results.  Arnaud's  (1889)  quantitative  colori- 
metric comparison  of  the  carotin  content  of  etiolated  and  green  leaves 
of  the  kidney  bean,  referred  to  above,  led  to  completely  opposite 
results.  Arnaud's  data  (calculated  from  his  colorimetric  reading) 
show  34.0  mg.  carotin  in  100  grams  of  the  dry  etiolated  leaves,  and 
178.8  mg.  in  the  same  amount  of  dry  green  leaves,  a  result  which 
appears  to  have  been  substantiated  by  the  observation  of  Schunck  on 
etiolated  and  green  daffodil  leaves.  Kohl  (1902f)  studied  the  same 
question  and  drew  the  same  conclusion  as  did  Arnaud,  namely,  that 
carotin  increases  during  greening.  His  method  of  analysis,  however, 
does  not  permit  so  exact  an  interpretation,  for  he  merely  compared 
colorimetrically  the  total  unsaponifiable  pigment  extracted  from  the 
leaves  by  alcohol.  Kohl's  carotin  solutions  were  thus  a  mixture  of 
carotin  and  xanthophylls.  It  is  not  possible  to  decide  from  these 
observations  whether  xanthophylls  as  well  as  carotin  increase  in  the 
etiolated  leaf  during  greening.  This  appears  to  be  the  case,  however, 
in  view  of  Ewart's  (1918)  statement,  quoted  above,  and  the  fact  that 
xanthophylls  are  the  predominating  carotinoids  in  green  leaves  as 
found  by  Willstatter  and  Stoll  (1913)  and  Miss  Goerrig  (1917). 

An  interesting  phase  of  the  etiolated  leaf  pigmentation  is  that  of  the 
most  favorable  conditions  for  the  development  of  the  carotinoids. 
Light  and  temperature  are  obviously  the  controlling  factors.  Wiesner 
(1877b)  observed  that  potato  sprouts,  which  formed  in  the  light, 
showed  little  if  any  yellow  pigment,  while  those  which  formed  in  the 
dark  developed  from  30  to  150  per  cent  more  pigment.  More  interest- 
ing is  the  result  of  Elfving  (1882),  which  was  confirmed  by  Immen- 
dorff  (1889),  that  carotinoids  increased  greatly  in  leaves  under  con- 
ditions which  depressed  chlorophyll  formation,  i.e.,  low  temperatures 
(2°  to  8°  C.)  and  very  diffused  light. 

Carotinoids  in  Naturally  Yellow  Leaves 

Plastids  which  fail  to  develop  chlorophyll  but  in  which  other  pig- 
ments form  instead  are  called  chromoplastids.  The  pigments  of 
chromoplastids  are  usually  granular,  sometimes  crystalline  and  almost 
invariably  yellow  to  red  in  color.    In  the  case  of  some  plants  the  leaf 


54  CAROTINOIDS  AND  RELATED  PIGMENTS 

plastids  are  always  characterized  by  an  absence  of  chlorophyll,  the 
leaves  being  yellow  or  golden  yellow  in  color.  Several  investigators 
have  studied  the  pigmentation  of  such  plants  in  relation  to  the  yellow 
chromolipoids  which  characterize  the  chloroplastid.  Thudichum 
(1869),  many  years  ago,  observed  the  relation  between  the  pigment  of 
the  carrot  root  and  that  in  the  yellow  leaves  of  Coleus,  and  included 
them  both  in  his  group  of  luteins.  Dippel  (1878)  found  that  the 
spectroscopic  absorption  bands  of  the  pigment  extracted  by  alcohol 
from  yellow  leaves  corresponded  with  those  of  the  yellow  pigment 
which  he  found  to  be  present  in  Kraus'  cyanophyll  layer  from  green 
leaf  extracts.    Dippel  called  this  pigment  xanthin' 

Tammes  (1900)  and  Kohl  (1902g)  first  sought  to  show  the  relation 
of  the  pigment  of  such  leaves  to  carotinoids.  Tammes  found  that  the 
plastids  of  yellow  leaves  gave  positive  carotinoid  reactions  with  con. 
H2SO4,  con.  HNO3,  and  with  HCl  containing  phenol  and  with  bromine 
water,  when  the  leaves  were  first  dried.  Yellow  leaves  in  which  the 
chromoplastids  had  disintegrated  failed  to  give  these  reactions.  When 
examined  after  submitting  the  leaves  to  the  Molisch  alkali  crystalliza- 
tion method  brownish  yellow  crystals  of  various  shapes  were  observed 
in  each  of  the  following  cases: 

1.    Aucuba  japonica  Thunb.  (Japanese  Aokiba). 


jCILH^UjUU,    juyuil'tjl^u/     j.J.xuxai.^.     \.tj  a/^a,j^^;;cc    jr3,\ja.i.K/a,^  , 

Elaeagnus  latifolia  L. 

Euonymous  japonicus  L.  variety  sulphurea  (Japanese  spindle  tree). 

Sambucus  nigra  L.,  variety  aurea.  (European  elder). 


Kohl  (1902g)  substantiated  these  results,  also  using  the  Molisch 
method,  and  in  addition  obtained  carotinoids  in  the  following  plants 
with  naturally  yellow  leaves. 

1.  Abutilon  nervosum  (Flowering  maple). 

2.  Betula  species  (Birch). 

3.  Spiroea  species,  variety  aurea. 

Kohl  made  a  more  exhaustive  study  of  the  pigment  of  yellow  elder 
leaves  {Sambucus  nigra  foliis  luteis)  and  found  that  the  dilute  alco- 
holic KOH  extract  of  the  leaves  gave  up  only  a  small  part  of  the  pig- 
ment to  ether.  The  ether  extract  thus  obtained  gave  the  carotin 
spectrum,  but  the  pigment  remaining  in  the  alkaline  alcohol  layer 
showed  only  end  absorption  of  the  spectrum.  He  believed  that  this 
pigment  was  due  in  part  to  the  (3  xanthophyll  of  Tschirch  (1887),  and 
in  part  also  to  a  new  pigment  which  he  called  phyllofuscin,  which  dif- 
fers from  (3  xanthophyll  in  being  partly  extracted  from  its  aqueous 
solution  by  ether.     It  seems  likely  that  Kohl  was  dealing  here  with 


CAROTINOIDS  IN  THE  PHANEROGAMS  55 

Tswett's  (1908b)  group  of  colorless  chromogens,  whose  alkali  com- 
pounds are  characterized  by  a  dark  yellow  or  brown  color.  No  xan- 
thophyll  pigment  showing  spectroscopic  absorption  bands  is  present 
in  leaves  having  yellow  plastids,  according  to  Kohl. 

Van  Wisselingh  (1915)  made  some  microscopic  observations  of  the 
crystals  which  he  produced  in  the  yellow  spotted  leaves  of  Croton 
ovalfolius  Vahl.,  Graptophyllum  pictum  Griff,  (caricature  plant) ,  and 
Sambus  nigra  L.  foliis  var.,  using  both  the  alkali  method  .of  Molisch 
and  the  acid  method  of  Frank.  The  crystals  produced  by  the  latter 
method  are  described  by  van  Wisselingh  as  brown  crystal  aggregates, 
the  yellow  spots  in  the  leaves  of  Sambus  nigra  showing  reddish  colored 
crystals  also,  and  the  similar  spots  in  the  leaves  of  Graptophyllum.  pic- 
tum showing  orange-red  platelets  and  small  orange-yellow  crystal 
aggregates. 

Summarizing  our  knowledge  of  the  pigments  in  naturally  yellow 
or  yellow  spotted  leaves,  it  seems  certain  that  carotinoids  are  con- 
cerned in  part  in  the  pigmentation,  but  the  information  is  lacking 
regarding  the  types  and  distribution  of  the  carotinoids  between  carotin 
and  xanthophylls.  It  also  remains  to  be  determined  whether  water- 
soluble  pigments  of  the  type  whose  alkali  salts  are  golden  yellow  in 
color,  which  are  probably  related  to  flavones,  are,  as  Kohl  believed-, 
normally  a  part  of  the  pigmentation. 

Carotinoids  in  Yellow  Autumn  Leaves 

The  hidden  beauties  of  the  forest,  revealed  in  the  passing  of  the 
chlorophyll  of  the  leaves  in  the  autumn,  were  among  the  earliest  to 
attract  the  attention  of  the  plant  chemists.  The  author  has  not  had 
an  opportunity  to  determine  how  far  back  scientific  observation  on 
the  autumn  leaf  pigment  can  be  traced.  Guibourt,  however,  as  early 
as  1827,  expressed  the  belief  that  the  yellow  and  red  pigments  of 
autumn  leaves  were  due  to  a  coloring  matter  which  persisted  after 
the  green  chromula  of  the  leaves  had  disappeared.  Guibourt  observed 
that  those  families  whose  flowers  and  fruits  were  characterized  by 
yellow  pigments  had  yellow  leaves  in  the  autumn,  while  the  families 
with  red  colored  flowers  and  fruits  had  red  autumn  leaves. 

Macaire-Prinsep  (1828)  apparently  made  the  first  chemical  exami- 
nation of  the  autumn  pigments.  He  found  that  the  yellow  pigment  of 
the  autumn  leaves  of  the  Loijibardy  poplar  {Populus  fastigiata)  could 
be  extracted  with  ether  or  hot  alcohol;  that  the  pigment  thus  extracted 


56  CAROTINOIDS  AND  RELATED  PIGMENTS 

turned  green  on  treatment  with  alkalis;  that  a  yellow  autumn  leaf 
recovered  its  green  color  when  immersed  in  alkali,  while  green  leaves 
turned  yellow  and  finally  red  when  treated  with  acids.  He  accordingly 
proposed  the  term  polychrome  for  the  green  chromula  of  the  leaf 
which  thus  changes  from  green  to  yellow  to  red  and  vice  versa,  and 
regarded  the  phenomena  which  he  observed  as  the  exact  duplication 
of  the  yellow  and  red  autumn  coloration  of  leaves.  Berzelius  (1837a), 
a  little  later  extracted  the  yellow  color  from  the  autumn  leaves  of  the 
pear  tree  {Pyrus  communis)  with  alcohol  (about  85  per  cent),  and 
called  the  pigment  xanthophyll.  Berzelius  noticed  that  the  pigment 
readily  bleached,  but  regarded  it  as  a  fat  and  as  being  derived  from 
the  green  pigment  of  the  leaf.  He  was  careful  to  distinguish  (1837b) 
between  the  pigment  xanthophyll  and  the  red  pigment  which  could 
be  extracted  from  the  red  autumn  leaves  of  the  mountain  ash  [Sorhus 
aucuparia)  and  cherry  trees  {Prunus  cerasus),  and  from  the  red 
autumn  leaves  of  gooseberry  [Ribus  glossularia,  var.  rubra)  and  com- 
mon barberry  {Berberis  vulgaris)  bushes,  which  pigment  Berzelius 
called  erythrophyll. 

Following  these  very  earty  experiments  which  were  carried  out 
before  the  development  of  our  present-day  knowledge  of  the  yellow 
chromolipoids  one  finds  a  number  of  more  or  less  unrelated  observa- 
tions regarding  the  yellow  autumn  colors,  which  were  made  incidental 
to  some  of  the  plant  pigment  studies  which  have  already  been  reviewed 
in  connection  with  the  carotinoids  in  the  chloroplastid.  For  example, 
Fremy  (1860)  regarded  the  autumn  coloration  as  due  to  his  phylloxan- 
thine,  the  isolation  and  properties  of  which  were  described  in  an 
earlier  paragraph.  As  already  pointed  out,  Fremy  believed  that  two 
pigments  existed  in  green  leaves,  a  greenish-blue  phyllocyanine  and 
a  yellow  phylloxanthine,  and  that  the  autumn  colors  were  the  result 
of  the  fact  that  the  latter  pigment  was  more  stable  than  the  former. 
Sachs  (1863)  made  a  microscopic  study  of  leaves  during  the  autumn 
color  change  and  proposed  the  theory  that  the  chlorophyll  migrates 
out  of  the  plastids  leaving  behind  a  larger  number  of  intensely  yellow 
granules.  The  latter  were  actually  observed  and  were  soluble  in 
alcohol.  Mer  (1873),  however,  was  not  able  to  substantiate  this 
belief  regarding  a  migration  of  the  green  granules  out  of  the  leaf  cells, 
but  the  presence  of  yellow  or  brownish-yellow  plastids  in  the  cells  of 
the  autumn  colored  leaves,  at  least  in  the  necrobiotic  *  phase,  is  well 

*  According  to  Tswett  (1908  c)  the  autumn  coloration  occurs  in  two  phases,  namely, 
the  necrobiotic  and  the  postmortal.    The  former  is  always  characterized  by  a  yellowing 


CAROTINOIDS  IN  THE  PHANEROGAMS  57 

established  (Tammes  (1900),  Goerrig  (1917)).  True  chromoplastids 
are,  in  fact,  present. 

The  disappearance  of  chlorophyll  from  the  leaves  in  the  autumn  is 
alone  sufficient  evidence  of  vital  changes  taking  place  in  the  proto- 
plasm. Viewing  the  phenomenon  of  autumn  coloration,  therefore,  in 
the  light  of  what  is  now  known  regarding  the  yellow  chromolipoids  of 
the  chloroplastids  the  questions  naturally  raised,  as  recently  pointed 
out  by  Miss  Goerrig  (1917)  are:  (1)  are  the  autumn  pigments  merely 
the  yellow  carotinoids  already  present  in  the  chloroplastids,  (2)  are 
the  natural  carotinoids  of  the  green  plastids  augmented  or  possibly 
replaced  by  other  yellow  pigments  which  may  be  closely  related  but 
still  capable  of  being  differentiated  from  the  normal  carotinoids,  or  (3) 
are  the  autumn  pigments  entirely  new  substances  which  replace  the 
normal  carotinoids,  destroyed,  perhaps,  with  the  chlorophyll?  In  spite 
of  the  fact  that  these  questions  have  been  studied  by  Miss  Goerrig 
(1917)  and  by  Tswett  (1908b)  using  the  most  modern  methods,  the 
question  of  autumn  coloration,  at  least  with  respect  to  the  yellow 
colors,  is  not  yet  entirely  cleared  up.  One  can  apparently  state  defi- 
nitely that  the  yellow  colors  are  not  due  to  entirely  new  pigments. 
Whether  the  plastid  carotinoids  are  present  unchanged  or  slightly 
modified  is  not  so  certain.  It  is  also  uncertain  to  what  extent  yellow 
pigments  of  an  entirely  foreign  nature  are  present  and  what  part  they 
play,  if  any,  in  the  autumn  coloration/'  Tswett's  and  Miss  Goerrig's 
studies  differ  decidedly  on  this  point. 

We  know  from  Miss  Wheldale's  (1916)  splendid  monograph  that 
there  is  a  large  group  of  trees,  shrubs  and  climbing  plants  in  whose 
leaves  red  anthocyanins  form  abundantly  in  the  autumn.  Some  of 
those  mentioned  by  Miss  Wheldale  have  been  shown  by  certain  inves- 
tigators to  contain  carotinoids  also.  Other  plants  do  not  form  antho- 
cyanins in  their  foliage  in  the  autumn.  Miss  Wheldale  mentions  a 
number  of  the  latter  in  which  autumn  carotinoids  have  not  yet  been 
demonstrated.  These  three  groups  of  plants,  together  with  the  names 
of  the  investigators  who  have  demonstrated  carotinoids  in  the  autumn 
leaf,  are  summarized  in  Tables  1,  2  and  3. 

of  the  plastids  and  a  retention  of  the  osmotic  pressure  of  the  cell  plasma.  The  latter 
is  recognized  by  the  disappearance  of  the  plastid  pigments,  the  disintegration  of  the 
protoplasm  and  the  formation  of  brown,  reddish  brown  or  black  pigments  as  the  result 
of  an  oxidation  of  colorless,  water-soluble  chromogens. 

"In  isolated  cases  such  as  the  yellow  leaves  of  the  osage  orange  (Madura  auran- 
tiaca)  one  no  doubt  finds  an  abundance  of  the  characteristic  yellow  flavones,  morin  and 
maclaurin  found  in  the  wood  of  this  plant  (Kressmann,  1914)  in  addition  to  carotinoids 
(Goerrig,  1917). 


5B  CAROTINOIDS  AND  RELATED  PIGMENTS 

Table  1.    Autumn  Foliage  Containing  Both  Anthocyanins  and  Carotinoids 

■Acer  campestris  (Common  European  Maple),  Tammes,  Tswett,  Goerrig. 

Acer  platanoides  (Norway  Maple),  Tammes,  Tswett,  Goerrig. 

Acer  ■pseudo'platanus  (Sycamore),  Arnaud  in  green  leaves. 

Aesculus  Hippocastanum  (Buckeye),  Tammes,  Tswett,  Goerrig. 

Crataegus  pinnatifida  (Hawthorne),  Tswett. 

Hedera  helix  (English  Ivy),  Arnaud  in  green  leaves. 

Prunus  avium  (Sweet  Mazzard  Cherry),  Tammes. 

Pyrus  germanica  (German  Pear),  Tswett. 

Pyrus  ussuriensis  (Pear),  Tswett. 

Quercus  rubra  (Red  Oak),  Staats. 

Rosa  rugosa  (Rugosa  rose),  Tswett. 

Taxodium  distichum  (Bald  Cypress),  Goerrig. 

Vitis  Coignetiae  (Grape),  Goerrig. 

Table  2.    Autumn  Foliage  Containing  No  Anthocyanins 

Alnus  glutinosa  (Black  Alder) . 

Aralia  (Ginseng),  Tswett. 

Betula  alba  (White  Paper-birch),  Staats. 

Broussonetia  papyrifera  (Paper  Mulberry),  Goerrig. 

Carpinvs  Betulus  (European  Hornbeam). 

Castanea  (Chestnut). 

Celastrus  scandens  (Climbing  Bittersweet),  Tammes. 

Convalaria  majalis  (Lily-of-the- valley),  Tswett. 

Corylus  Avellana  (Hazelnut). 

Dioscora  batatas  decn.  (Yam),  Tammes. 

Fraxinus  excelsior  (Ash). 

Funkia  Sieboldii,  Tswett. 

Ginkgo  biloba  (Maidenhair  tree),  Tswett,  Goerrig. 

Gleditsia  triacanthos  (Honey  Locust),  Tswett. 

Iris  germania  (Fleur-de-lis  or  Iris),  Tswett. 

Juglans  regia  (English  Walnut). 

Larix  europaea  (European  larch),  Tswett. 

Lepidium  Draba  (Pepperwort),  Goerrig. 

Liriodendron  tulipijera  (Tulip  tree),  Tswett. 

Madura  aurantiaca  (Osage  (Drange),  Goerrig. 

Mirabilis  Jalapa  (Four-o'-clock),  Goerrig. 

Morus  alba  (Mulberry),  Arnaud  from  green  leaves. 

Platanus  orientalis  (Oriental  sycamore),  Goerrig. 

Polygonum  sachalinense  (Sacaline),  Goerrig. 

Populus  alba  (Silver  Poplar). 

Populus  nigra  (European  Black  Poplar). 

Populus  tremula  (Aspen  Poplar),  Tswett. 

Ptelea  trifoliata  (Hop  tree),  Tswett. 

Pyrv^  communis  (Common  Pear),  Berzelius. 

Quercus  (Oak),  except  Quercus  rubra  and  Q.  cocdnea. 

Quercus  Robur  (English  Oak),  Tammes. 

Rhu^  toxicodendron  (Poison  Ivy),  Tswett._ 

Salix  babylonica  (Weeping  Willow),  Goerrig. 

Salix  Caprea  (Goat  Willow),  Goerrig. 

Sparmania  ajricana,  Tswett. 

Vlrrms  campestris  (Elm),  Immendorff,  Tammes,  Goerrig. 

Table  3.    Red  Winter  Foliage  Due  to  Carotinoids 

Aloes,  Molisch. 
Conifers,  Lubimenko. 
Cryptomeria  japonica,  Tswett. 


CAROTINOIDS  IN  THE  PHANEROGAMS  59 

Cupressus  Naitnocki  (Cypress),  Tswett. 

Encephalartos  Hildebrandtii  A.  Br.  and  Bouche,  Lubimenko. 

Juniperus  virginiaca  (Red  Cedar),  Tswett. 

Macrozamia  species,  Lubimenko. 

Retinospora  plumosa  (Juvenile  Thuja),  Tswett. 

Selaginella  (Club  Moss),  Molisch,  Lubimenko. 

Taxics  baccata  (Yew),  Tswett. 

Thuja  orientalis  (Arbor  Vitse),  Tswett,  Monteverde  and  Lubimenko. 

Opinion  was  divided  even  among  the  older  investigators  as  to 
whether  one  or  several  pigments  are  involved  in  the  autumn  colora- 
tion, and  as  to  what  relation  they  bear  to  the  normal  pigments  of  the 
green  leaf.  Among  those  who  believed  that  only  one  pigment  is 
involved  may  be  mentioned  Pringsheim  (1874),  whose  spectroscopic 
observations  of  extracts  of  yellow  autumn  oleander  leaves  and  rye 
straw  showed  three  absorption  bands  in  the  blue  only.  Pringsheim 
concluded  that  the  pigment  involved  was  different  from  his  etiolin, 
and  he  adopted  Berzelius'  name  of  xanthophyll  for  the  yellow  autumn 
pigment.  Tschirch  (1884)  also  believed  that  only  one  pigment  exists 
in  yellow  autumn  leaves,  which  he  called  (3  xanthophyll,  to  distinguish 
it  from  an  a  xanthophyll,  the  yellow  pigment  in  green  leaves,  although 
he  regarded  the  two  xanthophylls  as  closely  related,  if  not  identical. 
Tschirch  believed  that  there  was  less  xanthophyll  in  autumn  leaves 
than  in  green  leaves.  The  pigment  thus  described  by  Tschirch  was 
probably  carotin.  Immendorff  (1889)  succeeded  in  obtaining  carotin 
crystals  from  alcoholic  extracts  of  the  yellow  autumn  leaves  of  the 
beech  (Fagus)  and  elm  (Ulmus  campestris) ,  and  although  he  admits 
that  he  secured  a  very  small  quantity  and  that  only  in  one  case,  his 
extracts  always  showed  the  carotin  spectrum,  which  caused  him  to 
conclude  that  carotin  is  the  cause  of  the  yellow  autumn  coloration. 
Tammes  (1900)  examined  the  fallen  autumn  leaves  of  a  number  of 
trees  and  shrubs  after  submitting  them  to  the  Molisch  alkali  crystal- 
lization method.  The  plants  examined  are  given  in  Tables  1  and  2. 
Carotinoid  crystals  were  observed  in  all  cases  in  which  the  leaves  still 
showed  the  presence  of  yellow  plastids.  Tammes'  conclusion  that  car- 
otin is  the  cause  of  the  yellow  autumn  coloration  is,  of  course,  not 
valid,  in  view  of  the  fact  that  we  now  know  that  the  Molisch  reaction 
is  not  specific  for  carotin. 

In  view  of  the  multiplicity  of  carotinoids  at  present  acknowledged 
to  exist  in  the  chloroplastids  the  idea  of  only  one  pigment  in  the  yellow 
autumn  coloration  is  not  acceptable.  A  number  of  the  older  investi- 
gators  concluded  that   more   than   one   pigment  was   involved   even 


60  CAROTINOIDS  AND  RELATED  PIGMENTS 

before  it  was  definitely  known  that  several  yellow  chromolipoids  exist 
in  the  green  leaf.  For  example,  Gregor  Kraus  (1872c)  believed  that 
the  yellow  autumn  pigment  was  due  in  part  to  his  yellow  xanthophyll 
and  in  part  to  a  yellow  water-soluble  pigment.  Sorby's  (1871a,  b) 
idea  that  autumn  coloration  is  due  to  varying  mixtures  of  xantho- 
phylls,  erythrophylls  and  chrysotannins  is  not  far  different  from 
Miss  Goerrig's  recent  conclusion  when  one  is  acquainted  with  the  par- 
ticular properties  of  Sorby's  pigment  groups.  Sorby's  xanthophylls 
are  our  present  carotinoids;  his  erythrophylls  are  acknowledged  to  be 
our  red  anthocyanins  (they  were  characterized  by  being  strikingly 
affected  in  color  by  alkalies  and  acids) ;  and  his  chrysotannins,  which 
he  believed  increased  during  the  autumnal  color  changes,  were  indefi- 
nite water-soluble  yellow  pigments  with  acid  properties  (related  to 
tannic  acid)  which  readily  deepened  in  color  on  oxidation.  Miss  Goer- 
rig  found  abundant  quantities  of  a  yellow  pigment  in  autumn  leaves, 
which  could  be  extracted  with  dilute  acetone.  Saponification  of  the 
extract  greatly  intensified  the  color,  and  the  unsaponified  pigment 
could  not  be  extracted  from  the  dilute  acetone  by  ether.  There  also 
seems  to  be  little  reason  to  doubt  the  identity  of  Sorby's  chrysotannin 
and  Miss  Goerrig's  unnamed  yellow  water-soluble  pigment  with  the 
so-called  autumn-xanthin  which  Staats  (1895)  extracted  with  alcohol 
from  the  yellow  autumn  leaves  of  the  linden  (Tilia),  beech  [Fagus], 
ash  (Fraxinus)  and  red  oak  {Quercus  rubra),  and  which  he  obtained 
in  the  form  of  a  red  crystalline  water-soluble  potassium  salt.  Staats 
ascribed  the  autumn  pigmentation  solution  to  this  coloring  matter, 
but  in  this  he  was,  of  course,  mistaken.  The  alcoholic  extract  of  the 
oak  leaves  first  turned  green  and  then  yellow  with  the  precipitation  of 
the  potassium  salt,  when  treated  with  KOH,  confirming  the  observa- 
tion of  Macaire-Prinsep  (1828),  mentioned  above,  on  extracts  from 
autumn  poplar  leaves.  The  explanation  of  this  interesting  color  reac- 
tion of  autumn  leaves  is  not  apparent. 

Carl  Kraus  (1875)  also  ascribed  the  autumn  coloration  to  more 
than  one  pigment,  naming  two,  xanthin,  especially,  and  also  xantho- 
phyll. To  explain  briefly  his  terminology  it  may  be  stated  that  his 
xanthophyll  was  practically  the  Gregor  Kraus  xanthophyll  in  that 
author's  alcohol-benzene  separation,  Carl  Kraus  characterizing  it 
further  because  of  its  change  to  a  blue  pigment  on  treatment  with 
acid  (this  is  either  the  phylloxanthine  reaction  of  Fremy  (1860),  or 
the  xanthophyll  (3  color  reaction  of  Tswett  (1911a)).  The  xanthin  of 
Carl  Kraus,  however,  is  undoubtedly  carotin,  since  he  found  it  in  the 


CAROTINOIDS  IN  THE  PHANEROGAMS  61 

benzene  layer  of  the  Gregor  Kraus  separation,  and  it  was  not  turned 
blue  by  acids. 

C.  A.  Schunck's  (1903)  splendid  spectroscopic  observations  of  the 
xanthophylls  included  an  examination  of  the  pigments  of  yellow 
autumn  leaves.  The  method  of  preparation  of  the  material  for  these 
studies,  which  has  already  been  described,  precludes  the  presence  of 
pigments  other  than  carotinoids  in  the  alcoholic  solution  submitted  to 
Schunck's  carbon  disulfide  separation.  Schunck  does  not  mention 
chrysophyll  (carotin)  in  connection  with  his  autumn  leaf  examination, 
but  his  xanthophyll  solution  gave  four  banded  spectra  in  practically 
all  cases.  The  conclusion  drawn  was  that  autumn  coloration  is  due 
to  L.  xanthophyll  and  a  preponderance  of  the  acid  derivative  of  B. 
xanthophyll,  which  is  characterized  by  a  four  banded  spectrum,  a 
result  which  strongly  supports  the  idea  that  certain  changes  do  occur 
among  the  carotinoids  of  the  leaf  during  the  necrobiotic  period. 

Kohl  (1902h)  made  a  careful  study  of  autumn  pigmentation  and 
ascribed  the  autumn  colors  to  carotin,  a  xanthophyll  (showing  a  four 
banded  spectrum  (see  Schunck) ) ,  (3  xanthophyll  (a  water  soluble  pig- 
ment with  no  spectroscopic  properties),  a  little  phyllofuscin  and  a 
small  amount  of  another  yellow  pigment  also  derived  from  chlorophyll, 
which  he  does  not  name.  One  must  agree  with  Tswett  (1908b),  how- 
ever, that  Kohl's  methods  are  open  to  serious  objection,  in  that  the 
preliminary  boiling  of  the  leaves  in  water,  before  the  extraction  of  the 
pigments  with  hot  alcoholic  potash  undoubtedly  brought  about  serious 
decompositions  because  of  the  high  acidity  of  the  cell  sap  in  autumn 
leaves.  Nevertheless,  Kohl's  observations  do  indicate  that  carotinoids 
may  be  expected  to  decline  noticeably  during  the  autumn  color  change, 
thus  confirming  the  belief  expressed  by  Tschirch  (1884).  Kohl  states 
that  it  is  sometimes  difficult  to  demonstrate  the  presence  of  carotin 
(carotinoids)  at  all  in  autumn  leaves,  and  concludes  that  the  intense 
yellow  color  of  some  autumn  leaves  is  due  to  the  formation  of  other 
yellow  pigments. 

Tswett 's  (1908b)  study  of  the  pigments  of  yellow  autumn  foliage 
appears  to  be  the  most  reliable  which  we  have  available  at  present  on 
which  to  base  a  definite  knowledge  of  the  autumn  yellow  colors.  It 
is  true  that  Miss  Goerrig's  (1917)  more  recent  study  gave  quite  oppos- 
ing results  in  some  particulars,  but  the  apparent  contradictions  are 
not  wholly  irreconcilable  when  one  takes  into  account  the  fact  that 
Tswett's  and  Miss  Goerrig's  studies  differ  in  at  least  two  very  sig- 
nificant points,  namely,    (1)   Tswett  studied  the  pigments  in  fresh 


62  CAROTINOIDS  AND  RELATED  PIGMENTS 

leaves,  while  Miss  Goerrig  first  dried  the  leaves  in  the  air  at  40°  C. 
(protected  of  course  from  the  light) ,  and  then  ground  them  to  a 
powder,  and  (2)  Tswett  submitted  his  pigments  to  confirmatory  tests 
for  the  various  carotinoids  (unfortunately  omitting,  however,  a  chro- 
matographic analysis) ,  while  Miss  Goerrig  drew  her  conclusions  solely 
from  a  Kraus  separation  between  alcohol  and  petroleum  ether..  The 
bearing  of  these  differences  in  procedure  on  the  conclusions  of  the  two 
investigators,  respectively,  will  be  pointed  out  below. 

Referring  first  to  Tswett's  investigation  one  finds  that  he  plucked 
the  autumn  yellow  leaves  of  19  different  plants  during  the  necrobiotic 
period,  and  macerated  them  thoroughly  with  glass  powder,  or  emery, 
and  MgO  (to  insure  the  absence  of  acids  in  the  extracts)  and  then 
extracted  the  pulp  with  petroleum  ether.  This  was  followed  by  an 
extraction  with  alcoholic  petroleum  ether.  The  latter  was  freed  from 
alcohol  by  washing  with  water  and  the  two  extracts  examined  sepa- 
rately both  spectroscopically  as  well  as  with  respect  to  their  adsorp- 
tion by  CaCOo,  and  also  as  to  their  separation  in  the  Kraus  proce- 
dure, using  80  per  cent  alcohol  and  petroleum  ether.  The  first  extrac- 
tion with  petroleum  ether  alone  should  extract  the  carotin,  if  present, 
and  the  second  extraction  with  alcoholic  petroleum  ether  should  re- 
move the  xanthophylls.  In  addition,  extractions  were  made  of  the 
macerated  leaves  with  alcohol  alone,  and  these  tested  in  the  Kraus 
system.  The  plants  examined  by  Tswett  may  be  found  by  referring 
to  Tables  1  and  2. 

The  result  of  this  study  was  to  show  that,  while  traces  of  chloro- 
phyllins  and  normal  carotinoids  were  present,  the  bulk  of  the  pigment 
of  the  yellow  autumn  leaves  examined  before  the  postmortal  period 
was  a  carotinoid-like  pigment  (or  group  of  pigments)  which  was 
almost  completely  adsorbed  from  petroleum  ether  by  CaCOg,  like  the 
xanthophylls.  At  the  same  time  the  pigment  was  epiphasic  like  caro- 
tin, i.e.,  found  in  the  petroleum  ether  layer  in  the  Kraus  separation,  in 
all  cases  except  the  extracts  *^  from  the  honey  locust  {Gleditsia  tria- 
canthos)  and  the  buckeye  {Aescidus  Hippocastanum) .  Spectroscopi- 
cally the  pigment  showed  three  absorption  bands  behind"  F,  but  as  their 
position  was  somewhat  variable  no  measurements  were  made.  Tswett 
called  the  pigments  (he  believed  a  mixture  to  be  present)  autumn 
xanthophylls.  Saponification  did  not  alter  their  carotin-like  property 
of  remaining  for  the  most  part  in  the  petroleum  ether  layer  in  the 

« The  hypopbasic  portions  of  these  extracts  were  unfortunately  not  examined  further 
by  Tswett. 


CAROTINOIDS  IN  THE  PHANEROGAMS  63 

Kraus  separation.  In  the  author's  opinion  the  pigments  might  better 
have  been  called  autumn  carotins,  for  the  behavior  of  the  carotinoids 
in  the  Kraus  separation  unquestionably  depends  primarily  upon  chemi- 
cal composition,  as  Tswett  (1911b)  himself  has  pointed  out,  while 
their  relation  to  adsorbents  is  largely  a  colloidal  phenomenon,  as 
already  explained,  and  is  not  necessarily  related  to  chemical  com- 
position, 

Tswett  also  made  a  careful  study  of  the  question  of  the  alleged 
(Sorby,  1871a,  b,  Kraus,  1872,  Kohl,  1902)  presence  of  water-soluble 
yellow  pigments  in  yellow  autumn  foliage,  with  most  convincing  re- 
sults. He  found  that  hot  water  decoctions  of  autumn  leaves,  obtained 
with  the  exclusions  of  as  much  air  as  possible,  were  scarcely  colored 
at  all,  and  that  similar  decoctions  with  dilute  acetic  acid  were  pale 
yellow,  while  extractions  with  alkaline  water  were  golden  yellow  to 
brown  or  reddish  brown.  The  more  golden  colors  were  destroyed  by 
acid,  but  the  deeper  ones  persisted.  Decoctions  with  water  slightly 
alkaline  with  carbonates  were  likewise  golden  yellow,  and  the  extracts 
acted  towards  acids,  alkalies  and  air  like  the  extracts  with  distilled 
water,  Tswett  obtained  further  proof  of  the  presence  of  colorless 
water-  and  alcohol-soluble  chromogens  '^  in  autumn  leaves  (he  regards 
the  chromogens  as  present  in  green  leaves  also),  which  give  golden 
yellow  salts  with  alkalies  and  oxidize  to  a  dark  brown  color,  by  shak- 
ing the  diluted  alcoholic  extract  of  the  yellow  tulip  and  maple  leaves 
with  chloroform.  This  removed  the  color,  leaving  a  colorless  hydro- 
alcoholic  layer  which  acted  towards  alkalies  like  the  distilled  water 
decoctions  from  the  leaves. 

Tswett  holds,  apparently  rightly,  that  colored  salts  of  the  above 
mentioned  chromogens  may  at  times  play  a  part  in  the  coloration  of 
autumn  leaves  during  the  necrobiotic  period.  It  would  appear  that 
the  definiteness  of  the  relation  between  this  period  and  the  true  post- 
mortal period  of  the  leaf  is  the  important  factor  in  determining  this 
type  of  coloration  for  Tswett  believes  that  the  brown  oxidation  prod- 
ucts of  the  yellow  alkali  salts  of  the  colorless  chromogen  of  the  leaves 
play  the  chief  role  in  the  postmortal  coloration  of  leaves,  a  reaction 
no  doubt  accelerated  by  oxidizing  enzymes. 

Miss  Goerrig's  (1917)  recent  study  of  yellow  autumn  pigments  was 
an  attempt  to  determine  quantitatively  the  relation  of  the  carotinoids 
in  the  green  and  autumn  leaf,  just  before  and  during  the  necrobiotic 

^  These  substances  are  probably  flavones  which  are  characterized  especially  by  their 
yellow  color  reaction  with  alkalies. 


64  CAROTINOIDS  AND  RELATED  PIGMENTS 

phases,  determining  colorimetrically  carotin  and  xanthophylls  (as  a 
group)  by  the  Willstatter  and  Stoll  (1913c)  method,  using  potassium 
dichromate  as  standard.  The  study  was  necessarily  most  exacting  and 
laborious.  In  general,  the  data  show  that  the  amount  of  autumn 
carotinoids  in  comparison  with  the  carotinoids  in  the  green  leaves  just 
before  the  necrobiosis  varies  with  the  kind  of  plant  and  the  character 
of  the  weather  during  the  latter  period,  sometimes  being  more  and 
sometimes  less,  but  that  the  autumn  carotinoids,  even  when  higher 
than  the  prenecrosis  pigments,  never  equal  quantitatively  those  pres- 
ent in  the  leaf  in  midsummer.  In  all  cases  the  xanthophylls  exceed 
the  carotin.  The  reader  is  referred  to  the  original  paper  for  other 
details.    The  plants  examined  are  noted  in  Tables  1  and  2. 

From  a  qualitative  standpoint  Miss  Goerrig's  results  differ  signifi- 
cantly from  Tswett's  in  two  particulars,  (1)  the  former  denies  the 
existence  in  autumn  yellow  leaves  of  carotinoids  differing  from  the 
normal  plastid  carotinoids,  (2)  and  finds  water-soluble  pigments  in 
abundance  so  that,  "When  one  compares  the  color  of  the  extracted 
meal  and  the  wash  waters  with  the  meal  before  extraction  a  good  idea 
is  obtained  of  the  frequently  small  significance  of  carotin  and  xan- 
thophylls in  the  autumn  leaf  pigmentation." 

With  regard  to  the  first  difference.  Miss  Goerrig's  conclusion  is  cer- 
tainly open  to  criticism  in  that  she  did  not  submit  her  carotin  and 
xanthophyll  fractions,  obtained  by  separation  between  alcohol  and 
petroleum  ether,  to  any  confirmatory  tests  whatsoever.  That  Miss 
Goerrig's  carotinoids  from  autumn  leaves  were  probably  the  same  as 
the  mixture  which  Tswett  calls  autumn  xanthophylls  is  indicated  by 
the  very  significant  statement  that  repeated  extractions  of  the  petro- 
leum ether  solutions  with  high  percentage  methyl  alcohol  were  fre- 
quently required  to  separate  the  xanthophylls  from  the  carotin.  It  is 
not  difficult  to  conjecture  that  such  xanthophylls,  like  Tswett's  autumn 
pigments,  would  be  mostly  epiphasic  between  petroleum  ether  and 
80  per  cent  alcohol,  the  normal  xanthophyll  solvent  in  the  Kraus 
separation. 

The  difference  between  Miss  Goerrig's  and  Tswett's  results  respect- 
ing water-soluble  pigments  can  not  be  explained  so  readily,  inasmuch 
as  the  former  proved  that  her  leaf  preparations  from  both  green  and 
autumn  leaves  did  yield  strongly  colored  extracts  to  both  distilled 
water  and  to  tap  water,  as  well  as  to  very  dilute  acetone.  Leaf  pow- 
ders from  leaves  dried  at  40°  C.  were  used  for  these  tests,  while  Tswett 
examined  only  the  fresh  tissues.     This  difference  alone  may  be  suf- 


CAROTINOIDS  IN  THE  PHANEROGAMS  65 

ficient  to  account  for  the  divergence  of  the  results  on  this  point.    This 
possibility  should  be  investigated. 

Carotinoids  in  Autumn  and  Winter  Reddening 

The  winter  reddening  of  leaves  of  the  English  ivy  (Hedera  helix) , 
privet  (Ligustrum  vulgare)  and  other  evergreens,  and  also  that  of  cer- 
tain herbaceous  plants  like  Saxifraga  umbrosa,  which  retain  their 
leaves  in  winter,  as  well  as  the  autumn  reddening  of  the  Rosaceae  and 
many  other  individuals  of  various  plant  orders  is  due  to  anthocyanin 
formation.  The  chemistry  of  autumn  and  winter  reddening,  there- 
fore, does  not  seem  to  fall  within  the  scope  of  this  monograph.  How- 
ever, from  the  investigations  of  Molisch  (1902),  Tswett  (1911b),  and 
Monteverde  and  Lubimenko  (1913b),  carotinoids  with  an  antho- 
cyanin-like  color  are  responsible  in  some  cases  for  autumn  and  winter 
reddening. 

Schimper  (1885)  first  examined  the  red  pigment  in  winter  foliage  of 
various  firs  (Abies)  and  other  conifers,  such  as  Thuja  ericoides,  Thuja 
standishi,  and  the  common  box  tree  [Buxus  sempervirens)  and  found 
it  soluble  in  alcohol,  benzene  and  CSg,  and  that  it  was  extracted  from 
alcohol  by  the  last  named  solvent.  He  also  noticed  the  red  pigment 
in  various  parts  of  the  plant  of  many  varieties  of  aloes,  e.g..  Aloe  ver- 
rucosa. Courchet  (1888)  later  investigated  the  properties  of  this  red 
pigment  and  concluded  that  it  was  different  from  carotin  and  other 
pigments  because  it  did  not  give  the  blue  color  reaction  with  con. 
H2SO4.  Molisch  (1902)  believed  that  he  was  dealing  with  the  same 
pigment  when  he  found  that  the  red  plastids  in  winter-red  foliage 
leaves  of  many  (26)  varieties  of  aloes  responded,  in  part  at  least,  to 
his  alkali  crystallization  method.  The  pigment  crystallized  in  the 
form  of  needles,  platelets,  prisms  or  little  stars,  with  a  garnet  red  or 
yellowish  brown  color,  and  gave  the  usual  reaction  with  con.  H^SO^, 
HNO3,  6tc.  Similar  crystals  were  obtained  even  more  abundantly  in 
the  chromoplastids  of  several  varieties  of  Selaginella.  Molisch  con- 
cluded that  the  winter  reddening  of  aloes  and  Selaginella  is  due  in 
part  to  a  red  carotin  (carotinoid)  .^ 

Tswett  (1911b)  likewise  has  found  a  red  carotinoid,  undoubtedly  the 
same  pigment,  in  the  winter  foliage  of  several  plants.  These  are 
enumerated  in  Table  3.     He  proposes  the  name  thujorhordin  for  the 

«  Molisch  found  a  similar,  if  not  the  same  pigment,  normally  imbedded  in  a  colorless 
stroma  in  the  common  horsetail  (Equisetum  arvense),  the  pigment  itself  having  been 
observed  first  by  Schimper  (1885). 


60  CAROTINOIDS  AND  RELATED  PIGMENTS 

pigment  because  he  first  isolated  it  from  arbor  vitse  {Thuja  orien- 
talis).  The  pigment  is  described  by  Tswett  as  being  ruby  red  in  car- 
bon disulfide,  rose  in  alcohol  and  yellow  in  petroleum  ether.  It  is  not 
adsorbed  to  any  extent  by  CaCOg  from  petroleum  ether  or  CSg  solu- 
tion^ but  is  almost  wholly  hypophasic  in  the  separation  between  petro- 
leum ether  and  80  per  cent  alcohol.  Tswett's  pigment  showed  three 
absorption  bands  in  petroleum  ether  and  four  in  carbon  disulfide. 

Spectroscopically  the  pigment  differs  from  any  previously  known 
carotinoid,  but  Tswett's  classification  of  the  pigment  as  a  carotin  is 
subject  to  the  same  criticism  as  his  classification  of  the  autumn  yellow 
pigments  as  xanthophylls.  It  has  already  been  pointed  out  that  chemi- 
cal composition,  not  color  and  colloidal  and  other  physical  and 
physico-chemical  properties,  must  be  the  correct  basis  for  the  classi- 
fication of  carotinoids.  The  hypophasic  relations  of  thujorhordin  in 
the  Kraus  separation  would  classify  it  as  a  xanthophyll.  Monteverde 
and  Lubimenko  (1913b)  and  Lubimenko  (1914a,  and  b)  have,  in  fact, 
already  classified  it  thus  under  the  name  rhodoxanthin,  which  appears 
to  have  been  the  name  first  applied  to  this  pigment  by  Tswett  (1910b). 
The  discovery  of  the  same  pigment  in  the  pond-weed  {Potamogeton 
natans)  by  Monteverde  and  Lubimenko  has  been  mentioned  in  an 
earlier  paragraph,  and  its  characteristic  properties  given. 

Monteverde  and  Lubimenko's  study  of  the  Thuja  pigment  differs 
from  Tswett's  in  that  the  pigment  was  isolated  in  nearly  pure  crystal- 
line form,  making  it  possible  to  show  that  Tswett's  four  banded  spec- 
trum for  rhodoxanthin  in  carbon  disulfide  was  probably  due  to  some 
admixed  carotin  or  xanthophyll,  the  pure  pigment  showing  only  three 
bands.  According  to  Monteverde  and  Lubimenko  many  of  the  coni- 
fers owe  their  winter  reddening  to  rhodoxanthin. 

Lycopin,  the  red  isomer  of  carotin,  is  also  involved  in  winter  red- 
dening, according  to  Lubimenko  (1914a).  Two  conifers  whose  cone 
scales  owe  their  winter  color  to  this  pigment  are  mentioned  in  Table  3. 
The  plants  were  studied  under  tropical  conditions. 

Carotinoids  in  Flowers 

Yellow,  orange  and  orange-red  tints  are  especially  abundant  among 
flowers.  Marquart  (1835)  was  the  first  to  name  the  yellow  flower  pig- 
ments, calling  them  anthoxanthins  to  distinguish  them  from  the  blue, 
violet  and  red  pigments  which  he  called  anthocyanins.  Marquart 
noticed  that  certain  of  the  anthoxanthins  gave  a  blue  color  with  con. 


CAROTINOIDS  IN  THE  PHANEROGAMS  67 

H2SO4,  so  that  observations  regarding  flower  carotinoids  may  right- 
fully be  said  to  have  begun  with  this  investigator. 

Beginning  with  Fremy  and  Cloez  (1854),  however,  it  has  been  rec- 
ognized that  yellow  flower  colors  may  be  divided,  at  least  roughly,  into 
two  groups,  one  insoluble  in  water  and  the  other  soluble  in  water. 
Fremy  and  Cloez  called  the  former  xanthin,  and  regarded  the  pigment 
of  the  sunflower  (Helianthus  annus)  as  the  type.  The  pigments  of 
the  water-soluble  group  were  called  xanthein,  and  the  yellow  pigment 
which  may  be  obtained  from  certain  dahlias  was  regarded  as  the  type. 
Fremy  and  Cloez's  xanthin  was,  of  course,  a  mixture  of  carotinoids, 
and  their  xantheins  are  recognized  today  as  anthocyanins  and  flavones, 
only  a  few  of  which,  however,  have  been  isolated  and  closely  studied. 
We  are  concerned  in  this  monograph  only  with  the  carotinoids,  but 
the  subject  of  yellow  flower  pigments  is  somewhat  complicated  because 
of  the  fact  that  yellow,  orange  and  orange-red  pigments  of  a  consti- 
tution entirely  foreign  to  that  of  the  carotinoids  are  frequently  the 
cause  of  the  color  of  flowers  and  sometimes  associated  with  the  caro- 
tinoids in  causing  the  coloration. 

It  does  not  appear  to  be  possible  to  determine  with  certainty  by 
mere  inspection  whether  a  yellow  colored  flower  owes  its  color  to 
carotinoids  or  to  pigments  of  the  water-soluble  group,  although  Bid- 
good  (1905)  states  that  in  general  all  floral  colors  of  a  primrose  or 
sulfur-yellow  color  are  produced  by  the  latter  pigments,  and  that  such 
flowers  have  a  more  delicate,  transparent  appearance.  Microscopic 
observation,  however,  readily  reveals  the  character  of  the  pigment 
present,  for  the  carotinoids  appear  to  be  always  present  in  flowers  in 
the  form  of  chromoplastids,  while  the  anthocyanins  and  flavones  are 
always  present  in  solution  in  the  cell  sap.  There  may  be  some  excep- 
tions to  both  statements  but  the  differentiation  is  sufficiently  general 
to  serve  as  the  basis  for  determining  the  character  of  the  flower  color- 
ation. Confirmatory  tests  for  carotinoids  can,  of  course,  always  be 
carried  out.  Yellow  and  orange  colored  anthocyanins,  giving  a  red 
color  with  sulphuric  acid,  are  more  common  in  flowers  than  are  flav- 
ones, according  to  Bidgood,  who  lists  a  number  of  flowers  whose  color 
is  due  to  the  former,  but  very  few  that  owe  their  tints  to  the  latter. 

The  vast  majority  of  yellow  to  orange-red  colored  flowers,  however, 
owe  their  color  to  carotinoid  containing  chromoplastids.  Thudichum 
(1869)  gave  a  list  of  32  flowers  whose  yellow  pigment  he  regarded  as 
due  to  lutein.  Carotinoids  have  since  been  demonstrated  in  practi- 
cally all  these  cases.    Gregor  Kraus  (1872)  first  noticed  the  spectro- 


68  CAROTINOWS  AND  RELATED  PIGMENTS 

scopic  similarity  of  alcoholic  extracts  of  yellow  flowers  with  the  xan- 
thophyll  which  he  obtained  from  green  leaves  by  his  well-known 
method  of  separation  of  the  green  and  yellow  chloroplastid  pigments, 
Sorby  (1873)  believed  that  yellow  and  orange  colored  flowers  were 
colored  by  a  mixture  of  the  various  xanthophylls  and  xanthines  which 
he  described  and  which  have  already  been  reviewed  in  detail.  Dippel 
(1878)  also  noticed  the  similarity  between  the  absorption  spectra  of 
flower  extracts  (he  used  the  golden-yellow  flowers  of  the  California 
poppy  {Eschscholtzia  californica)  and  the  yellow  pigments  of  chloro- 
plastids.  Hansen  (1884c)  apparently  obtained  the  first  crystals  of 
carotinoid  from  yellow  flowers,  but  their  probable  impurity  is  indicated 
by  their  ease  of  solubility  in  both  alcohol  and  petroleum  ether,  crys- 
talline carotin  being  practically  insoluble  in  the  former  and  crystal- 
line xanthophylls  in  the  latter  solvent.  Hansen  called  the  flower 
plastid  pigment  lipochrome,  because  of  its  similarity  in  properties  to 
the  animal  lipochromes  which  were  being  studied  by  Krukenberg 
(1880-1886)    about  that  time. 

Schimper  (1883,  1885)  first  observed  that  the  orange-yellow  pig- 
ments in  the  chromoplastids  of  certain  flowers  existed  naturally  in 
crystalline  condition,  while  in  others  the  pigment  was  granular  or 
amorphous.  These  observations  were  greatly  extended  by  Courchet 
(1888),  who  used  the  name  chromoleucites  first  proposed  by  van 
Tieghem  in  place  of  chromoplastids.  The  former  has  not  had  such 
general  use  as  the  latter.  Courchet's  extensive  investigation  was  not 
confined  to  the  yellow  and  orange  pigments  of  flowers  and  fruits,  but 
covered  the  anthocyanins  as  well.  He  described  very  minutely  the 
morphology  and  organization  of  the  chromoplastids  and  the  pigments 
contained  therein  for  many  flowers  and  fruits.  He  found  a  very  inter- 
esting relation  to  exist  between  the  color  of  a  pigment  and  the  form 
which  it  assumes  in  the  plant.  He  showed  clearly  how  to  distinguish 
between  red  anthocyanin  pigments  dissolved  in  the  cell  sap  and  red 
plastid  pigments  frequently  deposited  in  crystals  in  the  plastids,  the 
latter  being  characterized  by  their  blue  color  reaction  with  con. 
H2SO4 ;  in  form  and  color  and  reactions  with  reagents  they  were  shown 
to  be  identical  with  the  red  crystals  in  tomato  plastids,  which  were 
believed  at  that  time  to  be  identical  with  the  carotin  of  carrots.  He 
demonstrated  that  yellow-orange  and  red-orange  colors  in  flowers  are 
always  found  in  the  chromoplastids,  frequently  in  the  form  of  crystals 
whose  rhombohedral  platelets  or  prisms  were  recognized  as  extraordi- 
narily closely  related  to  the  carotin  of  carrots  and  green  leaves.    He 


CAROTINOIDS  IN  THE  PHANEROGAMS  69 

pointed  out  distinctly  the  difference  between  yellow  flower  pigments 
dissolved  in  the  cell  sap  and  yellow  flower  pigments  deposited,  appar- 
ently always,  in  amorphous  condition  in  the  plastids.  Courchet  was 
successful  in  recrystallizing  the  red,  red-orange  and  yellow-orange 
plastid  pigments,  but  not  the  yellow  plastid  coloring  matters.  The 
latter,  however,  gave  the  same  color  reactions  with  the  lipochrome 
reagents  as  the  crystallizable  pigments,  and  their  great  solubility  in 
absolute  alcohol  shows  clearly  that  they  are  to  be  classified  as  xan- 
thophylls.  It  is  not  so  certain  that  the  crystalline  forms  were  carotin 
in  all  cases  on  account  of  their  somewhat  variable  solubility  in  alcohol. 

Following  Courchet,  carotin  crystals  were  obtained  by  Immendorff 
(1889)  from  extracts  of  the  flowers  of  various  members  of  the  butter- 
cup {Ranunculus)  family  and  from  various  members  of  the  hawkbit 
(Leontedon)  family,  probably  especially  the  so-called  autumn  dande- 
lion {Leontedon  autumnale).  Mobius  (1885)  had  already  shown  that 
the  peculiar  oily  appearance  of  the  yellow  buttercups,  which  is  the 
cause  of  their  common  name,  is  caused  by  the  pigment  being  dissolved 
in  the  cells  in  oil  and  not  present,  as  usual,  in  plastid  form.  This 
flower  therefore  appears  to  present  an  exception  to  the  general  rule 
that  flower  carotinoids  are  present  in  chromoplastids.  In  this  con- 
nection, the^idea  of  Hilger  (1894)  and  his  pupils  Wirth  (1891)  and 
Kirchner  (1892),  that  the  carotin  pigment  of  the  pot  marigold  flower 
[Calendula  officinalis)  is  a  mixture  of  sterol  esters  of  various  fatty 
acids  and  an  unnamed  colorless  hydrocarbon,  has  been  completely  dis- 
proved. Pabst  (1892)  proposed  the  same  idea  to  explain  the  consti- 
tution of  the  pigment  of  the  red  pepper  {Capsicum  annum)  and  Ehr- 
ing  (1896)  the  pigment  of  the  tomato  {Lycopersicum  esculentum). 
Even  if  these  authors  had  regarded  carotin  as  a  sterol-like  substance 
in  union  with  fatty  acids,  which  does  not  appear  to  have  been  the 
case,  the  hydrocarbon  character  of  carotin  and  the  fact  that  xantho- 
phylls  apparently  contain  no  hydroxyl  group  would  render  their  con- 
clusion untenable. 

We  are  dependent  for  our  present  knowledge  of  the  distribution  of 
carotinoids  among  flowers  upon  the  observations  of  Tammes  (1900), 
Kohl  (1902),  Schunck  (1903),  Tschirch  (1904),  Bidgood  (1905)  and 
van  Wisselingh  (1915).  Tammes,  Kohl  and  van  Wisselingh  sub- 
mitted the  flowers  examined  by  them  to  one  or  more  of  the  micro- 
chemical  crystallization  methods  previously  described,  the  last  named 
investigator  supplementing  these  in  certain  instances  with  additional 
tests  on  the  crystals  thus  formed  in  order  to  determine,  if  possible. 


70  CAROTINOIDS  AND  RELATED  PIGMENTS 

whether  the  crystals  were  carotin  or  xanthophylls.  Schunck's  com- 
bined separation  and  photospectrographic  procedure,  together  with  the 
effect  of  certain  reagents  on  the  absorption  bands,  has  already  been 
described  in  detail.  Schunck  reported  especially  the  distribution  of 
his  L.  B.  and  Y.  xanthophylls,  respectively,  in  a  number  of  common 
yellow  flowers.  The  author  is  of  the  opinion  that  Schunck's  work  can 
be  relied  on  merely  as  having  shown,  that  xanthophyll  carotinoids  are 
present  in  the  particular  flowers  examined  by  him.  The  author  re- 
cently ^  sought  to  verify  Schunck's  observation  that  the  pigment  of 
the  common  dandelion  {Taraxacum  officinale)  is  compared  solely  of 
B.  xanthophyll,  which  corresponds  with  Tswett's  a  and  a^  xantho- 
phylls, with  the  view  of  determining  the  influence  of  a  single  xantho- 
phyll on  the  coloration  of  egg  yolk  when  fed  to  laying  hens.  Only  the 
purest  yellow  parts  of  the  dandelion  head  were  examined.  Applying 
relative  solubility,  spectroscopic  and  chromatographic  adsorption 
methods  to  the  extracted  pigment,  it  was  found,  however,  that  carotin 
and  at  least  three  xanthophylls  were  present,  carotin  being  especially 
abundant.  Xanthophyll  (3  (Schunck's  Y.  xanthophyll),  characterized 
by  the  peacock-blue  color  of  its  alcoholic  solution  on  treatment  with 
HCl,  was  among  the  xanthophylls  present. 

Tschirch  used  a  still  different  method  for  his  flower  studies.  The 
alcoholic  extracts  were  submitted  to  the  capillary  analysis  procedure 
first  introduced  by  Goppelsroeder  (1901),  and  the  carotinoid  char- 
acter of  the  principal  yellow  zone  confirmed  spectroscopically. 

This  capillary  method  of  separation  has  not  been  mentioned  pre- 
viously so  that  a  few  statements  concerning  its  character  may  be  made 
at  this  point.  The  alcoholic  extract  of  the  flower  whose  pigments  are 
to  be  examined  is  placed  in  a  cylindrical  vessel  with  a  flat  bottom  and 
strips  of  thick,  fat-free  paper,  such  as  that  used  for  the  Adams'  milk- 
fat  analysis,  are  immersed  in  the  solution  to  a  depth  of  about  one 
centimeter.  The  strips  used  by  Tschirch  were  5  cm.  wide,  18  cm.  long 
and  about  1  mm.  thick.  These  strips  are  hung  from  a  support.  Dur- 
ing the  course  of  several  hours  the  pigmented  extract  gradually  rises 
on  the  paper  and  as  it  does  so  differentiates  itself  into  colored  zones, 
strikingly  similar  in  appearance  to  those  obtained  in  the  Tswett 
chromatographic  analysis.  When  the  capillary  rise  has  ceased  the 
paper  strips  are  removed,  dried,  and  the  various  colored  zones  sepa- 
rated with  the  scissors.  The  pigment  in  the  individual  zones  is  puri- 
fied by  repeating  the  capillarity  until  the  paper  takes  up  only  one 

"  Unpublished  investigation. 


CAROTINOIDS  IN  THE  PHANEROGAMS  71 

pigment.  The  method  is  strikingly  pretty  but  has  the  serious  objec- 
tion that  the  various  carotinoids  are  likely  to  lose  some  of  their  char- 
acteristic properties  through  oxidation  before  a  pure  pigment  is  ob- 
tained. According  to  Tswett  (1906c),  the  differentiation  into  zones 
is  not  due  to  adsorption,  as  in  his  method,  but  merely  to  a  combined 
effect  of  surface  evaporation  and  precipitation  of  pigments  having 
varying  degrees  of  insolubility  in  the  increasingly  dilute  alcohol.  It 
is  difficult  to  believe,  however,  that  colloidal  adsorption  does  not  play 
a  part  in  this  phenomenon. 

Bidgood's  addition  to  the  subject  of  flower  pigments  is  a  list  of 
flowers  whose  orange,  brown  and  green  tones  are  due  to  the  combined 
effect  of  carotinoids  and  crimson  and  blue  anthocyanins. 

The  author  has  attempted  to  collect  in  Tables  4,  5,  6  and  7  the 
results  of  the  work  of  the  various  investigators  mentioned.  It  is  seen 
that  a  very  large  number  of  yellow  flowers  owe  their  color  wholly  or 
in  part  to  carotinoids.  It  should  be  understood  that  the  tables  include 
only  those  which  have  been  studied,  and  that  the  lists  are  not  neces- 
sarily complete.  A  few  statements  may  be  necessary  in  explanation 
of  the  separate  tables.  The  carotin-containing  flowers  in  Table  4 
must  not  be  regarded  as  containing  this  pigment  only.  As  a  matter 
of  fact  carotin  apparently  never  exists  alone,  at  least  in  flowers  and 
leaves,  although  it  may  be  the  predominating  pigment,  as  in  the 
corona  of  the  poet's  Narcissus,  and  the  daffodil  where  it  is  found 
already  crystallized  in  the  plastids  (Courchet,  Bidgood,  van  Wissel- 
ingh).  The  orange  colored  pigment  of  the  pollen  of  the  so-called 
mullein  {Verbascum  thapsiforma  L.)  appears  to  consist  wholly  of 
carotin  (Bertrand  and  Poirault,  1892).  The  flowers  which  are  starred 
in  Table  4  were  placed  there  largely  because  they  have  been  found 
to  yield  microchemical  crystals  by  the  acid  method,  which  may,  at 
least  provisionally,  be  regarded  as  specific  for  carotin  on  account  of 
the  great  sensitiveness  of  xanthophylls  toward  acid.  The  presence  of 
carotin  in  the  other  flowers  in  Table  4  has  been  substantiated  by  other 
observations.  Arnaud  determined  quantitatively  that  the  flower  petals 
of  the  sweet  violet  {Viola  odorata)  contain  124  mg.  carotin  per  100 
gms.  of  dried  petals.  The  xanthophyll-containing  flowers  of  Table  5, 
similarly,  do  not  necessarily  contain  this  class  of  carotinoids  only, 
although  it  is  possible  that  this  may  be  the  case  for  those  which  are 
starred,  judging  from  van  Wisselingh's  study  of  this  question.  This 
investigator's  conclusion  that  the  flowers  which  are  double  starred  in 
the  table  contain  only  one  xanthophyll  should  be  substantiated  by 


72  CAROTINOIDS  AND  RELATED  PIGMENTS 

chromatographic  analysis.  The  collection  of  flowers  in  Table  6  sig- 
nifies that  we  know  as  yet  only  that  carotinoids  are  present  in  these 
flowers.  Whether  the  color  effects  are  also  due  in  part  to  yellow  hued 
non-carotinoids  is  not  known.  The  cases  where  this  fact  appears  to 
have  been  established  are  reported  in  Table  7,  which  includes  as  well 
a  few  of  the  known  cases  where  other  color  tones  are  due  in  part  to 
carotinoids. 

To  summarize  the  whole  subject  of  flower  carotinoids  very  briefly, 
it  is  clear  that  much  work  remains  to  be  done  before  our  knowledge 
can  be  regarded  as  complete  regarding  the  character  and  distribution 
of  the  individual  carotinoids  among  the  yellow  flowers. 

Table  4.    Yellow  Flowers  in  Which  Carotins  Have  Been  Demonstrated 

*Abutilon  Darwinii  Hook  (Flowering  Maple),  Tammes. 

Aloe  verrucosa,  Molisch. 

Asclepias  Cwassavica  L.  (Milk  weed),  van  Wisselingh. 

Asphodeliis  cerasifer  L.  (Asphodel),  Courchet. 
^Calceolaria  rugosa  Hook  (Lady-slippers),  van  Wisselingh. 
*Chelidonium  majus  L.  (Celandine  Poppy),  Tammes. 

Isatis  Tinctoria  L.  (Dyer's  Woad),  van  Wisselingh. 

Liriodendron  tulipifera  (Tulip  tree),  Schrotter-Kristelli. 
*Manettia  bicolor  Paxt.,  Tammes. 

Momordica  balsamina  (Balsam  Apple),  G.  and  F.  Tobler. 

Narcissus  poeticus  L.  (Poet's  Narcissus),  Courchet,  Bidgood,  van  Wisselingh. 

Narcissus  Pseudo-narcissus  L.  (Daffodil),  van  Wisselingh. 
*Nonnea  lutea  D.  C,  Tammes. 
*Priniida  officinalis  (Cowslip),  Tammes. 
*Siphocampylos  bicolor  G.  Dow,  Tammes. 
*Stilophorum  diphyllum  Nutt.,  Tammes. 

Taraxacum  officinale  Weber  (Common  Dandelion),  Palmer. 
*Trollius  asiaticus  L.  (Globe  Flower),  Tammes. 

*  Carotin  by  the  acid  microchemical  crystallization  method. 

Table  5.    Yellow  Flowers  in  Which  Xanthophylls  Have 
Beibn  Demonstrated 

Calceolaria  (Lady-slippers),  Schunck. 
**Calendula  arvensis  L.,  van  Wisselingh. 

Calendula  officinale  L.  (Pot  Marigold),  Schunck. 

Cheiranthus  cheira  L.  (Wall-flower),  Schunck. 
**Chelidonium  majus  L.  (Celandine  Poppy),  van  Wisselingh. 

Chrysanthemum  (probably  jrutescens  L.)  (Marguerite),  Schunck. 

Cytisus  Laburnum  L.  (Broom  Flower),  Schunck. 
*Dendrobium  thyrsiflorum  Rchb.  fil.  (Orchid),  van  Wisselingh. 
**Doronicum  Pardalianches  L.  (Leopard's  Bane),  Schunck,  van  Wisselingh. 
*Gazania  splendens  Hort.,  Kohl,  van  Wisselingh. 

Helianthus  annus  (Sunflower),  Schunck. 
**Hieraceum.  aurantiacum  L.  (Orange  Hawkweed,  or  Devil's  Bit),  van  Wisselingh. 

Isatis  tinctoria  L.  (Dyer's  Woad),  van  Wisselingh. 
**Lilium  croceum  Chaix.,  van  Wisselingh. 

Mimulus  moschatus  L.  (Musk  plant),  Schunck. 

*  Flowers  containing  xanthophylls  and  no  carotin,   according   to   van   Wisselingh. 

**  Flowers   whose    pigmentation   is   due   to    one   xanthophyll    only,    according    to    van 
Wisselingh. 


CAROTINOIDS  IN  THE  PHANEROGAMS  73 

Narcissus  Pseudo-narcissus  L.  (Daffodil),  Schunck,  van  Wisselingh. 

Primula  officinalis  (Cowslip),  Kohl. 

Ranunculus  acris  L.  (Buttercup),  Kohl,  Schunck. 

Raphanus  raphanistrum  L.  (White  Charlock),  Schunck. 

Ribes  aureum  (Golden  Current),  Kohl. 

**Spartium  junceum  L.  (Spanish  Broom),  van  Wisselingh. 

Tagetes  erecta  (Marigold),  Schunck. 

Taraxacum  officinale  Weber  (Common  Dandelion),  Schunck,  Palmer. 

Tropaeolum,  m,ajus  (Nasturtium),  Schunck. 

Tulipa  Gesneriana  L.  (Common  garden  Tulip),  van  Wisselingh. 

Tussilago  Farfara  L.  (Coltsfoot),  Schunck. 

Ulex  europoeus  (Gorse),  Schunck. 

Verbascum  species  (Mullein),  Kohl.  • 

Viola  tricolor  L.  (Pansy),  Schunck. 

**  Flowers   whose   pigmentation    is   due   to    one   xanthoptiyll   only,    according   to    van 
Wisselingh. 


Table  6.    Yellow  Flowers  in  Which  Carotinoids  Have  Been  Demonstrated 

Abutilon  megopotamicum  (Flowering  Maple),  Tammes. 

Abutilon  nervosum  (Flowering  Maple),  Kohl. 

Adonis  vernalis  (Spring  Adonis),  Tammes. 

Alyssum  saxatile  (Golden-tuft),  Tammes. 

Aster  species,  Courchet. 

Buphthalmum  salicifolium,  Tschirch. 

Cacalia  coccinea,  Tschirch. 

Caltha  palustris  (Marsh  Marigold),  Kohl,  Tammes,  Tschirch. 

Chrysanthemum  frutescens  (Marguerite),  van  Wisselingh. 

Clivia  miniata  Regel,  van  Wisselingh. 

Colutea  media  (Bladder  Senna),  Tschirch. 

Corydalis  lutea  D.  C.  (Lark's  Spur),  van  Wisselingh. 

Cucurbita  foetissima  (Calabazilla),  Kohl. 

Cucurbita  melanosperma  A.  Br.,  van  Wisselingh. 

Cytisus  Laburnum  L.  (Broom),  van  Wisselingh. 

Cytisvs  sagittalis  Koch.  (Broom),  van  Wisselingh. 

Doronicum  Columnae  Tenore  (Leopard's  Bane),  Kohl,  Tammes,  Tschirch. 

Doronicum  plantagineum  L.  excelsum,  van  Wisselingh. 

Doronicum  Pardalianches,  Tschirch. 

Epimedium  macrantheum,  Tammes. 

Eranthis  hyemalis  Salisb.  (Winter  Aconite),  Tammes,  van  Wisselingh. 

Erysimum  Perofskianum  Fisch.  and  Mey.,  van  Wisselingh. 

Ferula  species  (Giant  Fennel),  van  Wisselingh. 

Forsythia  Fortunei  (Golden  Bell),  Tammes. 

Forsythia  viridissima  (Golden  Bell),  Tammes,  Kohl,  Tschirch. 

Fritillaria  Imperialis  (Crown  Imperial),  Tammes,  Kohl,  van  Wisselingh. 

Gaillardia  splendens,  Tschirch. 

Gazania  species,  Tschirch. 

Genista  racemosa,  Tammes. 

Genista  tinctoria  (Dyer's  Greenwood),  Courchet. 

Geum  montanum,  Tschirch. 

Gongora  galeata  Reichb.,  van  Wisselingh. 

Helenium  autumnale  (Sneezewood),  Tammes. 

Hemerocallis  Middendirffii  Trautv.  and  Mey  (Yellow  Day  Lily),  van  Wisselingh. 

Hieracium  murorum  L.  (Hawkweed),  van  Wisselingh. 

Hieracium  Pilosella  (Mouse-ear  Hawkweed),  Courchet. 

Impatiens  Noli-tangere  (Touch-me-not),  Kohl,  Tammes. 

Inula  Helenium  L.  (Elecampane),  van  Wisselingh. 

Iris  Pseudacorus  L.,  van  Wisselingh. 


'  74  CAROTINOIDS  AND  RELATED  PIGMENTS 

Kerria  japonica  D.  C.  (Japanese  Rose),  Kohl,  Tammes,  Tschirch,  van  Wisselingh. 

Kleinia  Galpini  (Groundsel),  van  Wisselingh. 

Kniphofia  alooides  (Torch  Lily  or  Poker  Plant),  Tammes. 

Ladamun  hybridum,  Kohl. 

Leontedon  autumnalis  (Autumn  Dandelion),  Immendorff. 

Leontedon  Taraxicum  (probably  Common  Dandelion),  Tschirch. 

Lilium  bulbiferum,  Hansen. 

Loasa  lateritia,  Kohl. 

Lycaste  aromatica,  Tammes. 

Meconopsis  cambria  Vig.  (Welsh  Poppy),  van  Wisselingh. 

Melilotus  officinalis  (Sweet  Clover),  Tschirch. 

Nuphar  luteum  Sibth.  (European  Pond  Lily),  van  Wisselingh. 

Oenothera  biennis  (European  Evening  Primrose),  Tammes,  Kohl, 

Physalis  Franchetti  (Chinese  Lantern  Plant),  Tammes. 

Ranunculiis  auricomus  (Buttercup),  Tammes,  Kohl. 

Ranunculus  Ficaria,  Tammes,  Kohl. 

Ranunculus  Gramineus,  Tammes. 

Ranunculus  repens,  Kohl,  Tammes. 

Rosa  species,  yellow  flowers,  Hansen,  Kohl. 

Rudbeckia  Newmanii  (Cone  Flower),  Tammes. 

Silphium  perfoliatum  (Cup  plant).  Kohl. 

Sinapis  alba  L.,  van  Wisselingh.  ■  . 

Sisymbrium  Sophia,  Tammes. 

Strelitzia  Reginae  (Bird-of-Paradise  flower),  Tammes,  Kohl. 

Tagetes  patula  (Marigold),  Courchet,  Kohl,  Tammes. 

Telekia  speciosissima,  Tschirch. 

Thermopsis  lanceolata  R.  Br.,  van  Wisselingh. 

Tillandsia  splendens,  Tammes. 

Tritonia  aurea  (Blazing  Star),  Kohl,  Tschirch. 

Trollius  europaeus  (Globe  Flower),  Tammes,  Kohl. 

Tropaeolum  majus  (Nasturtium),  Courchet,  Tammes,  Kohl. 

Tropaeolum  minus  (Nasturtium),  Kohl,  A.  Meyer. 

Tulipa  Gesneriana  L.,  van  Wisselingh. 

Tulipa  hortensis  Gaertn.,  van  Wisselingh. 

Uvalaria  grandifiora  (Bellwort),  Tammes. 

Verbascum  Thapsiforma  (Mullein),  Tschirch. 

Viola  odorata  (Sweet  Violet),  Molisch,  Kohl. 

Viola  biflora,  Tschirch. 

Viola  cornuta  L.  var.  Daldowie  yellow  (Horned  Violet),  van  Wisselingh. 

Viola  lutea  (Yellow  petal  Violet),  Tschirch,  Tammes. 

Waldsteinia  geoides,  Tammes. 

Table  7.    Flowers  Containing  Carotinoids  and  Other  Pigments 

Allium  siculum  (Onion),  Courchet.     Chiefly  carotinoids  with  some  chlorophyll, 

giving  brown  color. 
Armeria  vulgaris   (Thrift),  Courchet.     Chiefly  carotinoids,  some  soluble  j^ellow 

non-carotin  oids. 
Atropa  belladona  (Belladonna),  Courchet.    Chiefly  carotinoids,  some  soluble  non- 

carotinoids. 
Bulbine  semibarbata,  Courchet.    Chiefly  carotinoids,  some  soluble  j^ellow  antho- 

cyanins. 
Crocus  sativum,  Tschirch.    Some  carotinoids  in  flower  petals,  also  Safran.    Pollen 

grains  Safran. 
Cypripedium  Boxollii,  C.  insigne,  C.  argus.  (Lady's  Slipper),  Bidgood.     Chloro- 
phylls back  of  chromoplastids  giving  brown  effect. 
Eschscholtzia  calif ornica  (California  Poppy),  Courchet.  Chiefly  anthocyanins,  with 

some  xanthophylls  (?). 
Geum  coccineum,  Courchet.    Partly  orange  colored  carotinoids  and  partly  orange 

colored  anthocyanin. 


CAROTINOIDS  IN  THE  PHANEROGAMS  75 

MaslevaUia  Veitchiana,  Bidgood.    Purple  cell  sap  and  carotinoid  chromoplastids. 
Narcissus  Tazetta  (Polyanthus  Narcissus),  Bidgood.    Chiefly  yellow  anthocyanin 

with  some  carotin  (?).  . 

Odintiglossunis    (Orchids),    Oncidiums    (Orchids),    Tropoeolums    (Nasturtiums), 

Bidgood.     Many  varieties  of  these  have  crimson  anthocyanin  in  epidermal 

cells  and  yellow  carotinoids  along  inner  walls  of  same  cells. 
Wallflowers,  Bidgood.    Some  have  crimson  sap  and  carotinoids  in  plastids. 
Yellow  tulips,  Bidgood.     Blue  anthocyanin  in  epidermal  cells,  overlying  yellow 

chromoplastids  in  staminal  filaments,  giving  green  effects. 

Carotinoids  in  Fruits 

A  large  number  of  yellow,  orange  and  red  colored  fruits  have  been 
examined  by  various  investigators  for  the  nature  of  the  pigment.  The 
coloring  principles  found  in  most  cases  can  be  classified  with  the  caroti- 
noids. For  the  majority  of  the  fruits,  however,  we  have  the  obser- 
vation of  only  one  investigator  and  this  in  many  cases  has  been  very 
inadequate  for  the  purpose  of  properly  classifying  the  kind  of  caroti- 
noid present.  Even  for  many  of  the  fruits  that  have  been  studied  by 
more  than  one  investigator  we  only  know,  as  yet,  that  carotinoids  are 
the  cause  of  the-  pigmentation.  There  is  great  need  for  an  application 
of  the  Tswett  system  of  analysis  to  the  pigments  of  these  fruits.  Only 
in  one  case,  namely,  the  tomato,  has  a  thorough  study  of  the  pigment 
been  made.    This  work  will  be  referred  to  in  detail  presently. 

The  fruits  for  which  only  a  single  observation  has  been  made  will 
be  reviewed  first,  briefly,  considering  the  cases  chronologically.  The 
various  fruits  for  which  more  than  one  observation  is  available  will 
be  considered  separately. 

Thudichum  (1869)  classified  the  pigment  of  the  fruit  of  Crataegus 
crus-galli  (Cockspur  thorn)  and  Cyphomandra  betacea  (Tree  tomato) 
as  luteins.  It  seems  reasonable  to  suspect  that  carotinoids  are  in- 
volved, but  nothing  further  is  known  regarding  their  character. 

Gregor  Kraus  (1872)  observed  orange-red,  round  or  spindle  forms 
in  the  fruit  flesh  of  Solanum  pseudo-capsicum  (Jerusalem  Cherry), 
but  the  pigment  involved,  which  is  obviously  a  carotinoid,  is  not 
known  further. 

Schimper  (1885)  observed  amorphous  red  and  orange-yellow  pig- 
ment forms  in  the  fruit  of  Bryonia  dioica  (Bryony)  and  red  amor- 
phous pigment  in  the  fruit  of  Loniceria  tataria  (Honeysuckle),  but 
these  pigments,  apparently  carotinoid  in  nature,  have  not  been  ex- 
amined in  detail. 

Courchet  (1888)  not  only  examined  the  character  of  the  pigment 
in  the  plastids  of  several  fruits  but  extracted  the  pigment  and  recrys- 


76  CAROTINOIDS  AND  RELATED  PIGMENTS 

tallized  it.  For  example,  carmine  colored  rhombohedral  crystals  with  a 
few  orange-red  trapezoidal  forms  were  obtained  from  the  ether  extract 
of  the  fruit  flesh  of  Cucumis  melo  (Muskmelon) .  In  the  case  of  the 
little  tomato-like  fruits  of  Eugenia  uniflora  (Pitanga  or  Surinam 
cherry)  a  red  anthocyanin  was  found  in  the  epidermis  cells,  and 
orange  colored  chromoplastids  in  the  pericarp  which  crystallized  in 
the  form  of  red-orange  rhombic  plates,  and  is  thus  obviously  caro- 
tinoid  in  nature.  The  ether  extract  of  the  berries  of  Douce-amere  and 
Solanum  corymbosum,  however,  deposited  both  a  yellow  amorphous 
xanthophyll-like  pigment  and  thin,  pale,  red,  rhombohedral  platelets, 
frequently  grouped  together  in  clusters.    The  latter  may  be  carotin. 

Desmouliere  (1902)  extracted  a  yellow  pigment  from  the  juice  of 
Prunus  armeniaca  (Apricot)  with  amyl  alcohol  and  the  residue  from 
this  solution  gave  the  lipochrome  reaction  with  con.  HgSO^.  He  con- 
cluded that  the  pigment  was  probably  carotin  but  there  is  no  evidence 
that  only  one  carotinoid  was  present.  The  character  of  the  pigment 
of  the  apricot  and  also  that  of  peaches  should  be  examined  in  the  light 
of  our  present  knowledge  of  the  carotinoids. 

Kohl  (1902)  obtained  carotinoid  crystals  by  the  Molisch  micro- 
chemical  crystallization  method  in  the  case  of  the  fruits  of  Berberis 
vulgaris  (Common  Barberry),  and  several  kinds  of  Olivias  and  Coton- 
easters,  but  his  conclusion  that  carotin  only  was  involved  we  know 
now  to  be  a  mistake. 

Tschirch  (1904)  made  a  capillary  colorimetric  analysis  of  the  alco- 
holic extract  of  the  little  fruits  of  Euonymous  europaeus  (European 
Spindle-tree)  and  obtained  several  yellow  to  red-orange  zones.  The 
chief  zone  of  the  latter  color  unquestionably  showed  the  spectrum  of 
carotin.    Whether  other  carotinoids  are  present  is  not  known. 

Duggar  (1913)  obtained  spectroscopic  and  physiological  evidence 
that  the  principal  pigment  of  the  carpellary  tissue  of  Momordica 
charantia  (Balsam  Pear)  is  carotin  but  that  lycopin,  the  red  tomato 
pigment,  characterizes  the  bright  red  aril  of  the  seed. 

Monteverde  and  Lubimenko  (1913b)  found  the  bright  red  pulp  of 
the  tropical  fruit  Trichosanthus  to  owe  its  pigment  to  lycopin  and 
carotin,  chiefly  the  former. 

Lubimenko  (1914a)  examined  the  pigment  of  a  large  number  of 
fruits  in  the  famous  botanical  gardens  at  Buitenzorg,  Java,  in  order 
to  determine  the  effects  of  the  tropical  conditions  on  the  development 
and  character  of  the  pigment.  The  predominating  pigment  found  was 
lycopin  or  a  closely  related  pigment  which  Lubimenko  calls  lycopin- 


CAROTINOIDS  IN  THE  PHANEROGAMS  77 

oid,  because  of  slight  differences  in  the  relative  intensity  of  the  first 
two  absorption  bands  as  compared  with  the  same  bands  of  lycopin, 
and  because  of  a  greater  ease  of  solubility  in  absolute  alcohol  and 
glacial  acetic  acid.  In  the  case  of  the  fruit  of  Arum  orientale,  the 
chief  pigments  were  carotin  and  xanthophylls,  true  lycopin  making  up 
only  a  small  part  of  the  pigment.  Various  Aglaonema  fruits,  such  as 
Ag.  rdtidum  Kunth,  Ag.  ohlongij olium  Kunth,  Ag.  oblong,  variety 
Curtisii,  and  Ag.  simplex  Bl.  were  found  to  contain  variable  amounts 
of  yellow  pigments  besides  lycopin.  The  following  fruits  appeared  to 
contain  chiefly  lycopin:  Actinophlocus  angustijolius  Becc,  Actino- 
phlocus  macarthurii  Becc,  Archonthophoendx  Alexandrae  H.  Wendl., 
Calyptrocalix  spicatus  Blume,  Erythroxylum  nova-granadense,  Nenga 
Schefferiana  Becc,  Nertera  depressa  Banks  and  Soland,  Pandanus 
polycephalus  Lam.,  Ptychandra  glauca  Scheff.,  Ptychosperma  elegans 
Blume,  Sinaspadix  Petrichiana  Hort.,  Solanum  decasepalum,  Tanerno- 
montana  pentastycha  Scheff.  Especially  interesting  were  the  fruits  of 
Gonocarium  obovatum  Hocr.  and  Gon.  pyriforme  Scheff.,  the  bands  of 
whose  pigment  in  carbon  disulfide  solution  were  intermediate  between 
the  characteristic  bands  of  carotin  and  lycopin.  Lubimenko  regarded 
the  lycopin  of  the  fruit  of  the  palm  Areca  Alicae  W.  Hill  as  a  lyco- 
pinoid  because  its  first  two  spectroscopic  absorption  bands  in  carbon 
disulfide  were  of  equal  intensity  while  the  second  band  in  the  case  of 
lycopin  is  more  intense  than  the  first. 

Van  Wisselingh  (1915)  obtained  a  positive  carotinoid  reaction  on 
the  fruits  of  Viburnum  Opulus  (European  cranberry  bush),  using  the 
Molisch  microchemical  crystallization  method,  but  made  no  further 
study  of  the  crystals. 

Gill  (1918)  has  found  carotinoids  in  palm  oil,  the  commercial  prod- 
uct which  is  obtained  from  the  fruits  of  certain  Palmacece,  particu- 
larly Eloeis  guineesis  L.  (Jacq.),  which  form  vast  forests  along  the 
West  Coast  of  Africa,  and  Eloeis  melanococca,  Garb.  (Alfonsia  olei- 
fera,  Humb.)  which  is  cultivated  in  the  West  Indies  and  in  South 
America.  Gill's  observations  are  of  interest  in  the  light  of  Lubi- 
menko's  study  of  the  pigments  of  the  palm  fruits,  mentioned  above. 

Asparagus  berries.  Thudichum  (1869)  classified  the  pigment  with 
the  luteins.  Hartsen  (1873b)  described  the  red  granules  of  pigment 
in  the  berries  and  stated  that  red-colored  crystalline  tablets  of  the 
pigment  were  insoluble  in  water,  soluble  in  alcohol  and  ether  and 
especially  so  in  petroleum  ether.     The  pigment  thus  appears  to  be 


78  CAROTINOIDS  AND  RELATED  PIGMENTS 

carotin,  but  whether  other  carotinoids  are  present  has  not  been  de- 
termined. 

Ampelopsis  hederacece.  Hansen  (1884)  classified  the  pigment  of 
the  fruit  as  a  lipochrome  and  Kohl  (1902)  obtained  carotinoid  crys- 
tals by  the  Molisch  microchemical  method, 

Aglaonema  commutatum  Shott.  Tammes  (1900)  obtained  positive 
carotinoid  color  tests  on  the  chromoplastids  of  the  fruit.  Van  Wissel- 
ingh  (1915)  made  a  detailed  study  of  the  microchemical  crystals 
obtained  by  the  Molisch  method  and  concluded  that  the  chief  pigment 
is  lycopin,  but  that  other  carotinoids  are  present  also. 

Citrus  limonum  (Lemon).  Neither  Kohl  (1902)  nor  Tschirch  (1904) 
could  find  evidence  of  carotinoids  being  involved  in  any  way  in  the 
pigmentation  of  the  yellow  skin  of  this  fruit. 

Cucumis  citrullis  (Watermelon).  A.  and  G.  de  Negri  (1879)  first 
isolated  the  pigment  of  the  flesh  of  this  fruit  and  called  it  rubidin 
because  of  its  red  color.  Red,  needle  shaped  crystals  were  described, 
soluble  in  ether,  benzine  and  chloroform,  forming  yellow  or  yellow- 
red  solutions,  and  in  carbon  disulfide  forming  a  magnificent  rose- 
colored  solution.  The  insolubility  of  the  crystals  in  alcohols  and  the 
characteristic  three-banded  absorption  spectrum  which  was  found  to 
be  identical  with  that  of  the  red  tomato  pigment  obviously  classifies 
the  pigment  as  a  carotin  if  not  as  lycopin  itself,  as  there  is  good  reason 
to  believe.  Courchet  (1888)  also  crystallized  the  watermelon  pig- 
ment and  found  that  the  crystals  resembled  completely  in  form  and 
in  color  those  obtained  from  the  tomato.  Monteverde  and  Lubimenko 
(1913b)  have  definitely  confirmed  its  identity  with  lycopin,  as  well 
as  to  show  that  carotin  and  xanthophyll  are  also  present  in  the  red 
fruit  pulp. 

Cucurbito  pepo  L.  (Pumpkin).  Arnaud  (1885)  stated  that  he  ob- 
tained crystals  of  pigment  from  the  flesh  of  this  fruit  which  were  iden- 
tical in  properties  with  carrot  carotin.  Schrotter-Kristelli  (1895b) 
later  made  a  closer  study  of  the  pigment  of  this  family  of  plants, 
using  for  the  source  of  his  material  the  thin  deep-red  outer  layer  of 
the  pericarp  of  so-called  Turk's-cap  gourd.  The  pigment  was  not 
found  to  be  readily  extractable  by  alcohol,  even  by  hot  absolute  alco- 
hol, but  was  readily  soluble  in  petroleum  ether,  ether,  chloroform  and 
carbon  disulfide.  The  recrystallized  pigment  was  found  to  be  iden- 
tical in  solubility  and  in  its  reactions  with  carotin,  especially  the 
emerald  green  color  on  addition  of  HCl  to  the  alcoholic  solution  of 
the  pigment.    The  conclusion  seems  justified  that  carotin  is  the  chief 


CAROTINOIDS  IN  THE  PHANEROGAMS  79 

pigment  of  the  fruits  of  the  gourd  family,  including  pumpkins  and  the 
various  yellow  fleshed  squash  varieties.  Whether  other  carotinoids 
are  also  present  has  not  been  determined. 

Gill  (1918)  has  recently  stated  that  carotin  is  found  in  yellow 
squash,  the  statement  being  based  on  the  use  of  the  Crampton- 
Simons  palm  oil  test  which  this  author  has  found  to  be  due  to  carotin 
(probably  carotinoids  in  general). 

Momordica  balsamina  (Balsam  Apple).  G.  and  F.  Tobler  (1910) 
first  studied  the  pigment  of  this  fruit  and  believed  that  two  pigments 
were  present;  a  yellow  one  soluble  in  alcohol,  ether,  benzene  and  fatty 
oils,  the  ether  solution  showing  the  absorption  bands,  478-465^.[x  and 
435-4:15\i\i;  a  ruby  red  pigment,  extractable  by  cold  alcohol,  but  not  by 
benzene,  but  soluble  in  both  of  these  solvents  as  well  as  in  ether, 
chloroform  and  fatty  oils,  which  failed  to  show  the  lipochrome  reac- 
tions with  H2SO4  and  I2-KI,  but  which  showed  the  following  four- 
banded  spectrum  in  benzene. 

I.  513-496[X!i;    II.  487-446[i}x;      III.  455-443fxn;     IV.  437-425txn. 

The  yellow  pigment  was  found  chiefly  in  the  exo-  and  mesocarp. 
Its  solubilities  and  spectra  indicate  that  it  is  a  xanthophyll.  The  red 
pigment  was  found  chiefly  in  the  endocarp.  Its  absorption  spectrum 
in  alcohol  resembles  closely  that  of  lycopin  but  the  other  properties 
are  at  variance.  Duggar  (1913)  also  examined  the  balsam  apple  pig- 
ment and  found  the  carpellary  tissues  to  be  yellow  to  orange  as  did 
the  Toblers,  and  the  aril  to  have  a  bright  red  color.  Duggar  regards 
the  latter  pigment  to  be  lycopin,  on  spectroscopic  grounds,  but  the 
failure  of  the  pigment  to  show  other  characteristic  carotinoid  prop- 
erties, as  found  by  the  Toblers,  remains  to  be  explained. 

Physalis  alkekenzi  (Strawberry  Tomato,  Winter  or  Bladder 
Cherry).  Thudichum  (1869)  classified  the  pigment  of  this  fruit  as  a 
lutein,  and  Tammes  (1900)  obtained  positive  carotinoid  color  reac- 
tions with  the  plastid  pigment.  Monteverde  and  Lubimenko  (1913b) 
regard  the  pigment  as  carotin,  but  differing  from  it  by  the  compara- 
tive intensity  of  the  absorption  bands.    They  call  it  carotin  B. 

Arum  italicum  (Wild  Ginger).  Schimper  (1885)  observed  red 
amorphous  pigment  in  the  plastids  of  the  berries  of  this  plant. 
Courchet  (1888)  also  observed  the  brick-red  plastids,  and  obtained 
red-orange  lamella  and  carmine-red  rhomboids  from  the  yellow-orange 
ether  extract.  Kohl  (1902)  secured  microchemical  carotinoid  crystals 
using  the  Molisch  method.    Carotin  seems  to  be  one  of  the  pigments 


80  CAROTINOIDS  AND  RELATED  PIGMENTS 

involved  here.  Whether  other  carotinoids  are  present  remains  to  be 
determined. 

Lonicera  xylosteum  (Honeysuckle).  Schimper  (1885)  stated  that 
he  observed  red  and  orange-yellow  crystals  in  the  plastids  of  the  fruit. 
Molisch  (1896)  and  Kohl  (1902)  obtained  microchemical  crystals  by 
the  alkali  method  of  the  former  worker.  Nothing  further  is  known 
regarding  the  carotinoids  present. 

Physalis  franchetti  (Chinese  Lantern  Plant).  Carotin  is  appar- 
ently the  chief  if  not  the  only  pigment  present  in  the  fruit  from  the 
observations  of  Tammes  (1900),  Tschirch  (1904)  and  van  Wisselingh 
(1915).  Tammes  obtained  splendid  microchemical  crystals  by  the 
acid  method.  Tschirch  found  only  one  characteristic  orange-colored 
zone  in  the  capillary  analysis  of  the  alcoholic  extract,  showing  the 
carotin  bands. 

The  crystals  which  van  Wisselingh  obtained  by  the  alkali  micro- 
chemical method  were  insoluble  in  phenol-glycerine,  a  property  which 
he  found  to  be  characteristic  of  carotins. 

Sorhus  aria,  Crantz  (White  Beam-tree).  Thudichum  (1869)  clas- 
sified the  pigment  of  the  fruit  among  the  luteins.  Tammes  (1900) 
obtained  carotinoid  color  tests  on  the  plastid  pigment.  Van  Wissel- 
ingh (1915)  found  three  types  of  crystals  in  the  fruit  wall  after  15 
months'  treatment  by  the  Molisch  microchemical  method;  (1)  thin 
orange-red  platelets,  often  parallelograms,  (2)  orange  crystal  bundles, 
and  (3)  orange-yellow  crystal  masses.  The  classification  of  the  caro- 
tinoids present  remains  to  be  made. 

Tamus  communis  (Black  Bryony).  Both  Hartsen  (1873)  and 
Courchet  (1888)  obtained  red  crystals  from  extracts  of  the  berries, 
but  did  not  name  the  pigment.  Van  Wisselingh  (1915)  has  made  a 
closer  study  and  obtained  microchemical  evidence  of  lycopin  and 
xanthophylls,  but  not  of  carotin  in  the  fruit.  An  analysis  of  the  pig- 
ments present  using  the  Tswett  (1911a)  procedure  would  be  of  value 
in  confirming  this  interesting  case  of  carotinoid  distribution. 

Rosa  species.  Both  Tammes  (1900)  and  Kohl  (1902)  demonstrated 
carotinoids  in  fruits  of  this  family,  the  former  using  both  colorimetric 
and  alkali  crystallization  methods  and  the  latter  the  microchemical 
crystallization  (alkali)  method  only  on  the  plastids.  The  dark-orange 
capillary  zone  which  Tschirch  (1904)  examined  showed  the  carotin 
spectrum,  using  the  fruit  skins  of  Rosa  canina  (Dog  Rose) ,  as  source 
of  his  material.  Monteverde  and  Lubimenko  (1913b),  however,  have 
isolated  lycopin  crystals  from  the  dried  fruit  pulp,  but  they  neverthe- 


CAROTINOIDS  IN  THE  PHANEROGAMS  81 

less  regard  it  as  a  minor  constituent  of  the  pigments  of  this  fruit. 
The  microchemical  Molisch  crystals  which  van  Wisselingh  (1915) 
obtained  from  the  orange  fruits  of  Rosa  rugosa  Thumb.  (Rugosa 
Rose)  dissolved  readily  in  phenol-glycerine,  which  is  indicative  of 
xanthophylls.  The  pigmentation  of  the  various  rose  fruits  thus  ap- 
pears to  vary. 

Sorbus  aucuparia  (European  Mountain  Ash).    Immendorff  (1889) 
believed  carotin  to  be  the  pigment  of  the  fruit  of  this  plant.     Both 
Tammes  and  Kohl   obtained  microchemical   crystals   by  the   alkali 
method,  and  van  Wisselingh  (1915)  by  the  acid  method  as  well.    The 
latter  investigator  made  a  closer  study  of  the  crj'stals  obtained  and 
found  red  and  orange-red  platelets  insoluble  in  phenol-glycerine,  and 
orange  and  yellow-orange  platelets  and  needles  which  dissolved  readily 
in  this  reagent.    Both  carotin  and  xanthophylls  appear  to  be  present, 
and  a  Tswett  chromatographic  analysis  of  the  mixed  pigments  would 
probably  give  the  characteristic  chloroplastid  display  of  carotinoids. 
Citrus  aurantium    (Orange).     Tammes    (1900)    obtained   positive 
carotinoid  color  reactions  on  the  skin  plastids  but  failed  to  secure 
crystals  after  a  short  (18  day)  submission  to  the  alkali  microchemical 
crystallization  method.     Kohl  examined  the  pigment  of  the  pericarp, 
and  found  only  spectroscopically  inert  pigment,  although  he  thought 
there  might  be   traces   of   carotin    (carotinoids)    present.     Schunck 
(1903)  studied  the  skin  pigment  of  several  varieties  of  oranges  and 
found   considerable    amounts   of   water-soluble    (anthocyanin?)    pig- 
ments, especially  in  the  red  skin  varieties    (Blood   orange,   Seville 
orange  and  Tangerines).     He  found,  however,  that  the  alcoholic  ex- 
tracts yielded  crystals  of  chrysophyll  (carotin)   and  showed  spectro- 
scopically the  presence  of  acid  derivatives  of  B.  and  Y.  xanthophylls. 
Tschirch  (1904)  also  obtained  proof  of  the  presence  of  water-soluble 
non-carotinoid  pigments  in  the  orange  skin.     His  spectroscopic  study 
of  the  principal  carotinoid  pigment,  secured  by  the  capillary  method, 
did  not  give  satisfactory  results.    Gill  (1918)  has  obtained  a  carotin 
(carotinoid)  test  with  orange  skin  extracts,  using  the  color  reaction 
mentioned  above.    A  more  exact  study  of  the  orange  pigments,  using 
chromatographic  and   solubility  methods,   as  well   as  the   improved 
microchemical  methods,  would  seem  desirable. 

Solanum  dulcamara  (Bittersweet).  Thudichum  (1869)  classified 
the  pigment  as  lutein.  Hartsen  (1873)  obtained  red  crystals  identical 
with  those  from  Tamus  communis  and  Asparagus  berries.  Schimper 
(1885)  observed  red  crystals  in  the  fruit  plastids,  and  Tammes  (1900) 


82  CAROTINOIDS  AND  RELATED  PIGMENTS 

crystallized  them  by  the  Molisch  method.  According  to  Lubimenko 
(1914a)  lycopin  is  the  chief  pigment  present.  Van  Wisselingh  (1915) 
obtained  crystals  of  pigment  by  the  acid  microchemical  method  as  well 
as  by  the  alkali  method,  and  on  further  study  concluded  that  lycopin 
is  the  chief  pigment,  but  that  another  orange-red  carotinoid  is  present 
also,  which  fails  to  react  towards  the  Ig-KI  reagent.  A  further  study 
of  the  latter  pigment,  which  van  Wisselingh  found  in  other  fruits  also, 
would  seem  to  be  desirable.  It  has  previously  been  considered  that 
the  frequent  failure  of  the  iodine  reaction  was  characteristic  of  the 
animal  lipochromes  only,  and  was,  in  fact,  one  point  of  difference 
between  the  plant  and  animal  lipochromes.  This  differentiation  seems 
to  break  down  in  the  light  of  van  Wisselingh's  results. 

Capsicum  annum  (Red  Pepper).  The  red  pepper  pigment  has 
interested  a  number  of  plant  biologists.  Thudichum  (1869)  first 
classed  it  with  the  luteins.  Pabst  (1892)  was  unable  to  identify  it 
spectroscopically  with  carotin.  Kohl  (1902)  regarded  the  pigment  as 
completely  identical  with  carotin,  but  in  this  he  was  mistaken,  for 
Tschirch  (1904)  recognized  the  close  relation  of  the  pepper  pigment 
spectrum  with  that  of  lycopin.  Duggar's  (1913)  spectroscopic  obser- 
vations led  him  to  conclude  that  lycopersicin  (lycopin)  is  the  pigment 
of  both  the  skin  and  flesh  of  the  red  pepper.  While  van  Wisselingh 
(1915)  obtained  a  positive  carotinoid  test  using  the  alkali  crystalliza- 
tion method,  he  does  not  classify  the  Capsicum  fruit  among  those  con- 
taining lycopin.  It  should  be  stated  that  the  measurements  of  the 
absorption  bands  of  lycopin,  given  by  Tschirch  (1904),  do  not  cor- 
respond exactly  with  the  lycopin  bands  (in  the  same  solvent)  given 
by  Willstatter  and  Escher  (1910).  Monteverde  and  Lubimenko 
(1913b)  found  the  red  pepper  pigment  to  be  spectroscopically  identi- 
cal with  lycopin  but  because  of  the  ease  of  solubility  of  the  crude 
pigment  in  alcohol,  in  opposition  to  the  usual  difficult  solubility  of 
lycopin  in  this  solvent,  they  have  named  it  lycopin  B. 

Lycopersicum  esculentum  (Tomato).  The  red  tomato  pigment  has 
been  by  far  the  most  extensively  studied  of  the  fruit  pigments  of  the 
carotinoid  class,  and  is  the  only  one,  in  fact,  for  which  we  possess  at 
present  definite  chemical  knowledge  that  it  is  not  identical  with  the 
usual  carotin  and  xanthophylls  of  the  chloroplastids. 

Millardet  (1876),  who  first  investigated  the  tomato  pigment,  recog- 
nized that  it  is  not  identical  with  the  orange  and  yellow  pigments 
which  characterize  other  fruits.  He  therefore  proposed  the  name 
solanorubin  for  the  pigment.    It  is  recognized  now  that  the  name  was 


CAROTINOIDS  IN  THE  PHANEROGAMS  83 

not  very  wisely  chosen.  The  name  proposed  for  the  pigment  by 
Schurick  (1903),  who  apparently  was  unfamiliar  with  Millardet's 
investigation,  namely,  lycopin,  is  more  generally  used  at  present,  espe- 
cially since  Willstatter  and  Escher  (1910)  adopted  it  in  their  thorough 
chemical  study  of  the  pigment.  Duggar  (1913)  has  offered  the  name 
lycopersicin  as  being  more  suitable,  but  in  spite  of  Duggar's  very  valid 
arguments  against  the  name  lycopin,  the  latter  appears  likely  to 
become  the  prevailing  term  for  the  coloring  matter. 

Millardet  not  only  obtained  the  tomato  pigment  in  crystalline  con- 
dition, but  also  observed  the  crystals  in  the  flesh  of  the  ripe  fruit. 
The  crystalline  pigment  was  described  by  him  as  being  insoluble  in 
water,  soluble  in  alcohol  at  higher  temperatures,  and  easily  soluble  in 
CS2,  CHCI3  and  benzene.  It  showed  a  characteristic  spectrum  in 
CS2,  showing  two  bands  in  the  green  at  B  and  F,  respectively,  and  a 
third  in  the  blue  between  F.  and  G.  It  readily  bleached  in  the  light. 
Millardet  believed  that  the  pigment  was  derived  from  chlorophyll,  but 
this  idea  has  long  since  been  abandoned. 

A.  and  G.  de  Negri  (1879)  regarded  the  tomato  pigment  as  iden- 
tical with  the  rubidin  which  they  isolated  from  the  watermelon. 
Schimper  (1885)  observed  the  red  crystals  in  the  ripe  tomato  fruit, 
as  did  also  Courchet  (1888).  Arnaud  (1885),  however,  following  his 
discovery  of  carotin  in  the  chloroplastids,  believed  the  tomato  pig- 
ment to  be  carotin.  Passerini  (1890)  followed  Arnaud  in  this  belief 
and  so  did  Ehring  (1896),  Tammes  (1900)  and  Kohl  (1902).  Zopf 
(1895),  however,  could  not  identify  it  spectroscopically  with  carotin. 
Schunck  (1903),  also,  found  the  red  tomato  pigment  to  have  a  char- 
acteristic absorption  spectrum.  Schunck  believed  it  to  be  a  distinct 
pigment,  different  from  carotin,  and,  as  previously  stated,  named  it 
lycopin.  The  same  pigment  is  found  in  the  leaves  of  the  tomato  plant, 
according  to  Montanari  (1904),  but  this  fact  has  not  been  reported 
by  other  investigators. 

The  first  hint  of  the  true  relation  of  lycopin  to  carotin  was  obtained, 
however,  by  Montanari,  who  submitted  the  pure  crystals  to  analysis 
for  the  first  time.  He  obtained  an  average  composition  of  C  =  88.14 
per  cent  and  H  —  10.88  per  cent,  which  he  regarded  as  correspond- 
ing sufficiently  well  to  the  Arnaud  formula  for  carotin,  CasHag,  which 
was  still  in  vogue  at  that  time.  Molecular  weight  determinations  in' 
benzene,  using  the  cryoscopic  method,  gave  values  of  635-650,  from 
which  fact  it  was  concluded  that  the  pigment  was  dicarotin,  or  CggH^^, 


84  CAROTINOIDS  AND  RELATED  PIGMENTS 

the  theoretical  molecular  weight  of  which  is  698.  A  melting  point  of 
173.7°  C.  (corrected)  was  found  for  the  crystals. 

From  the  work  of  Willstatter  and  Escher  (1910),  however,  it  is 
evident  that  lycopin  is  identical  in  general  composition  and  molecular 
weight  with  carotin,  differing  only  in  solubility  in  certain  solvents  and 
in  the  position  of  the  absorption  bands  and  in  the  form  of  the  crystals 
as  well  as  their  color  when  free  and  in  solution.  There  is  a  slight 
difference  in  melting  point  also,  lycopin  melting  between  168°  and 
169°  C,  while  carotin  melts  at  167.5°  to  168°  C.  The  conclusion  that 
lycopin  is  a  true  isomer  of  carotin  seems  entirely  justified. 

The  isolation  of  lycopin  was  carried  out  by  Willstatter  and  Escher 
on  their  usual  generous  scale,  starting  with  74  kg.  of  tomato  conserve, 
from  which  11  grams  of  pure  recrystallized  pigment  were  eventually 
obtained.  Crystals  of  carotin  were  also  obtained  in  small  amounts  as 
by-product,  showing  that  the  factor  for  yellowing  which  the  red 
tomato  possesses,  and  which  is  familiar  to  the  botanist,  is  due,  in 
part  at  least,  to  the  usual  carotin  of  the  chloroplastids. 

The  analyses  and  molecular  weight  determinations  carried  out  by 
Willstatter  and  Escher  on  the  pure  lycopin  crystals  were  in  excellent 
agreement  with  theoretical  composition  and  molecular  weight  of  caro- 
tin as  found  by  Willstatter  and  Mieg  (1907),  namely,  C4oH5a,  as 
shown  by  the  following  data. 

Mol.  Wt.  found  for 
Calculated  jor         Found  jor  lycopin.        Calculated  Mol.  lycopin. 

CmHrn  (Ave.  of  4  detns.)  Wt.  for  doH^e         (Ave.  of  7  detns.) 

C  =  89.48  C  =  89.36 

H  =  10.52  H  =  10.81  536  558 

The  characteristic  properties  of  lycopin  as  described  by  Willstatter 
and  Escher  may  be  summarized  briefly  as  follows.  The  crystals  are 
dull  brownish-red  to  carmine  colored  flakes  and  lack  the  metallic 
iridescence  of  carotin  and  xanthophyll  crystals.  The  solution  in  CSg 
retains  its  bluish-red  color  on  great  dilution,  and  while  the  ethereal 
and  alcoholic  solutions  are  yellow  they  have  a  somewhat  browner  tone 
than  carotin  or  xanthophyll  solutions.  The  solubility  of  the  lycopin 
crystals  in  the  usual  carotin  solvents,  namely,  ether,  petroleum  ether 
and  CS2,  is  somewhat  less  than  that  of  carotin,  and  it  is  even  more 
difficultly  soluble  in  hot  alcohol  than  pure  carotin.  An  iodine  addi- 
tion product  of  constant  composition  and  characteristic  form  could 
not  be  obtained,  the  product  being  amorphous  and  having  an  iodine 
content  of  34-37  per  cent.    Lycopin  readily  oxidizes  and  bleaches  like 


CAROTINOIDS  IN  THE  PHANEROGAMS  85 

the  other  carotinoids,  but  the  oxidized  product  nas  a  characteristic, 
different  odor  from  oxidized  carotin,  according  to  Willstatter  and 
Escher.  Especially  characteristic  is  the  position  of  the  absorption 
spectral  bands  of  lycopin,  particularly  in  CSg,  three  bands  being  vis- 
ible, the  second  of  which  nearly  occupies  the  space  between  the  first 
two  carotin  bands.  The  measurements  as  carried  out  by  Willstatter 
and  Escher  are  as  follows,  using  a  0.05  per  cent  solution  in  carbon 
disulfide.  The  figures  have  been  confirmed  completely  by  Monteverde 
and  Lubimenko  (1913b). 

10  mm.  layer.  20  mm.  layer.  40  mm.  layer. 

Band  I      :  554  — 540     ^m-  561  — 555      -536    \i\i  563  -533      .  .525  \x.\i 

Band  II     :  514  — 499.4    "  517.5    -498     "  525  -493      .  .483     " 

Band  III  :  479  .  .472       "  481.5 — 468     "  483  -462.5  .  .427-  " 

Note:    -  means  very  dark;  —  means  fairly  dark;  .  .  means  rather  weak. 

Since  Willstatter  and  Escher's  thorough  study  of  the  red  tomato 
pigment,  Duggar  (1913)  has  observed  that  green  tomato  fruits  ripened 
above  30°  C.  do  not  form  lycopin  but  only  carotin  (possibly  xantho- 
phylls  also),  producing  a  yellow  fruit,  but  that  the  induced  yellow 
fruits  form  lycopin  if  the  temperature  is  reduced  to  the  usual  ripen- 
ing temperatures,  namely,  20°  to  25°  C.  These  facts  are  of  special 
interest  to  the  plant  physiologist  and  geneticists. 

Of  particular  interest  from  the  standpoint  of  those  desiring  to  iden- 
tify the  presence  of  lycopin  in  other  fruits  and  plants  is  van  Wis- 
selingh's  (1915)  study  of  the  microchemical  crystallization  of  lycopin 
and  the  effect  of  various  reagents  on  the  crystals  thus  formed.  This 
investigator  finds  that  lycopin  does  not  readily  crystallize  in  the 
tomato  fruit  by  the  Molisch  method  at  room  temperature,  but  does  so 
more  readily  at  80°  C,  and  very  readily  at  140°  C,  using  a  10  per 
cent  solution  of  KOH  in  glycerin  instead  of  in  alcohol,  the  high  tem- 
perature, of  course,  making  the  use  of  alcohol  unfeasible.  The  other 
carotinoids  fail  to  crystallize  at  the  high  temperature.  The  lycopin 
crystals  which  form  have  a  reddish-violet  color  and  show  a  charac- 
teristic color  change  with  bromine  from  red-violet  to  blue-violet  to 
blue-green,  green,  yellow,  and  finally  colorless.  Like  carotin,  the 
microchemical  lycopin  crystals  are  insoluble  in  phenol-glycerin  (3 
parts  by  weight  of  phenol  crystals  and  one  part  by  weight  of  glycer- 
in). Van  Wisselingh  also  found  what  appeared  to  be  carotin  crystals 
in  the  tomato  fruit  after  carrying  out  the  Molisch  procedure  at  80°  C. 


86  CAROTINOIDS  AND  RELATED  PIGMENTS 

Carotinoids  in  Seeds  and  Grains 

The  wide  distribution  of  carotinoids  in  flowers  and  fruits,  as  re- 
vealed in  the  foregoing  paragraphs,  naturally  justifies  the  expectation 
that  the  same  pigments  should  be  found  in  seeds  and  especially  in  the 
grains  of  plants  of  the  grass  species  where  fruit  and  seed  are,  for 
practical  purposes,  one  and  the  same. 

Of  the  true  seeds  the  plant  biochemists  who  have  studied  pigments 
naturally  have  been  interested  especially  in  the  highly  pigmented 
endocarp  or  aril  which  characterizes  a  number  of  plants.  Several 
of  these  have  been  the  object  of  investigation. 

Courchet  (1888)  recrystallized  the  ether  extractable  pigment  of  the 
arils  of  Euonymous  japonicus  (Japanese  Spindle-tree),  Momordica 
Balsamina  (Balsam  Apple)  and  Passiflora  coerulea  (Passion  flower 
plant).  The  red-orange  rhombic  shaped  tablets  obtained  from  the 
aril  of  the  Spindle-tree  indicate  the  close  relation  of  the  pigment  to 
carotin,  while  the  carmine  colored  needles  which  Courchet  obtained 
from  the  bright  red  arils  of  the  other  two  plants  were  recognized  by 
him  as  being  identical  in  form  and  color  with  those  obtainable  from 
tomatoes  and  from  the  flesh  of  watermelons.  It  would  appear  that 
the  more  orange  colored  endocarps  owe  their  color  to  carotin  (and 
probably  xanthophylls)  while  those  of  a  more  distinct  red  color  are 
pigmented  by  lycopin.  This  supposition  is  borne  out  by  the  observa- 
tion of  Schrotter-Kristelli  (1895a),  who  found  the  orange  color  of  the 
aril  of  Afzelia  Cuazensis  to  be  due  to  carotin,  dissolved  in  a  thick 
orange-yellow  oil,  from  which  he  recovered  and  recrystallized  the  pig- 
ment after  saponification  of  the  oil.  The  Toblers  (1910a)  and  Duggar 
(1913)  have  confirmed  Courchet's  observation  that  the  red  pigment 
in  the  bright  red  aril  of  Momordica  Balsamina  is  lycopin.  Duggar  has 
observed  also  that  the  aril  pigment  of  Momordica  charantia  is  lyco- 
pin, which  fact  has  already  been  mentioned.  Lubimenko  (1914a)  be- 
lieves that  the  aril  of  Euonymous  Japonicus  owes  its  color  to  the  same 
pigment. 

There  is  less  certainty  regarding  the  character  of  the  carotinoid  in 
the  arils  of  some  of  the  other  plants.  Tammes  (1900)  obtained  caro- 
tinoid color  reactions  and  a  Molisch  microchemical  crystallization  of 
the  pigment  in  the  aril  of  Euonymous  latijolia  Scop.  (Spindle-tree), 
which  was  substantiated  completely  by  van  Wisselingh  (1915).  Both 
Kohl  (1902)  and  van  Wisselingh  (1915)  obtained  positive  carotinoid 
reactions  for  the  aril  of  Taxus  baccata  (Yew  tree) ,  but  according  to 


CAROTINOIDS  IN  THE  PHANEROGAMS  87 

Monteverde  and  Lubimenko  (1913b)  this  pigment  is  rhodoxanthin,  the 
red  isomer  of  xanthophyll.  True  carotinoids  are  not  present  in  the 
aril  of  Myristica  jragrans  Houtt.  (Nutmeg),  judging  from  Kohl's 
(1902)  classification  of  the  pigment  as  a  xanthophyll  showing  no 
spectroscopic  absorption  properties.  Lubimenko  (1914a),  however, 
reports  lycopin  in  the  aril  of  this  plant. 

Other  seeds  which  are  not  characterized  by  highly  pigmented  endo- 
carpellary  tissue  have  been  found  to  contain  carotinoids  although 
nothing  is  known  regarding  the  distribution  of  the  individual  caro- 
tinoids among  the  total  pigment.  These  seeds  are  characterized  by 
yielding  a  yellow  oil  on  pressure.  Gill  (1918)  has  tested  by  a  carotin- 
oid  color  test  flax  seed  {Linium  usitatissimum,  the  linseed  of  com- 
merce), mustard  seed  {Brassica  nigra)  and  sesame  seed  {Sesamum 
indicum),  obtaining  a  positive  test;  and  rape  seed  {Brassica  campes- 
tris),  white  sunflower  seed  {Helianthus)  ,'^^  and.  cotton  seed  [Gossy- 
pium  hirsutum),  with  negative  results.  Palmer  and  Kempster  (1919c), 
however,  have  found  that  rape  seed  increases  slightly  the  color  of  the 
egg  yolk  when  fed  to  laying  hens,  indicating  the  presence  of  some 
xanthophyll  in  the  seeds.  Refined,  but  unbleached,  cottonseed  oil  is 
characterized  by  a  rich  golden  color  and  Palmer's  (1914g)  study  of 
the  character  of  the  pigments  of  cottonseed  meal  has  shown  that  this 
color  is  due  to  a  mixture  of  carotin  and  xanthophylls.  Hemp  seed 
{Cannabis  sativa)  was  found  by  Palmer  and  Kempster  (1919c)  to 
slightly  increase  the  color  of  egg  yolk,  and  thus  appears  to  contain 
xanthophyll  in  small  amounts. 

The  cereal  grains  also  appear  to  contain  carotinoids  more  or  less 
abundantly.  Thudichum  (1869)  classified  the  pigment  of  yellow 
Indian  corn  {Zea  mays)  with  the  luteins.  The  author's  (1914g)  study 
of  this  pigment,  however,  shows  it  to  be  almost  entirely  xanthophyll, 
with  a  little  carotin.  Spectroscopically  the  xanthophyll  corresponded 
with  the  principal  xanthophyll  (probably  xanthophyll  a)  of  the  chloro- 
plastids,  but  its  relative  solubility  and  adsorption  properties  were  at 
variance  in  that  it  did  not  seem  to  be  adsorbed  to  any  extent  from 
petroleum  ether  or  carbon  disulfide  by  CaCOg,  and  it  appeared  to  be 
just  as  readily  extracted  from  80  per  cent  alcohol  by  petroleum  ether 
as  from  the  latter  solvent  by  fresh  80  per  cent  alcohol.    The  peculiari- 

"  The  particular  variety  of  sunflower  seed  examined  is  not  clear.  The  common  sun- 
flower {Helianthus  annus)  whose  seed  is  used  for  commercial  oil  production,  gives  a 
very  pale  yellow  or  greenish-yellow  oil  and  it  is  possible  that  carotinoids  may  not  be 
present. 


88  CAROTINOIDS  AND  RELATED  PIGMENTS 

ties  of  this  pigment  have  not  yet  been  explained  and  the  work  should 
be  repeated. 

Monnier-Williams  (1912)  has  shown  that  carotin  is  one,  if  not  the 
chief  pigment  of  unbleached  wheat  flour.  The  author  has  confirmed 
the  presence  of  both  carotin  and  xanthophylls  in  the  wheat  grain 
(Triticum  vulgare).  Carotinoids  are  also  present  in  small  amounts  in 
barley  {Hordeum  sativum)  and  oat  {Avena  sativa)  grains,  as  shown 
by  Palmer  and  Kempster  (1919c)  and  even  in  traces  in  the  grains  of 
polished  rice  {Oryza  sativa) ,  as  shown  by  the  experiments  of  the  same 
authors  (1919a). 

Summary 

Carotin,  the  specific  pigment  of  the  carrot  root,  was  first  isolated 
and  named  by  Wachenroder  (1826).  The  hydrocarbon  nature  of  the 
pigment  was  discovered  by  Zeise  (1847)  and  confirmed  by  Arnaud 
(1886).  The  formula  C40H56  was  established  for  the  pigment  by 
Willstatter  and  Mieg  (1907)  and  confirmed  by  Euler  and  Nordenson 
(1908)  and  others.  Euler  and  Nordenson  showed  that  xanthophylls 
are  also  present  in  carrots,  a  fact  confirmed  by  Palmer  and  Eckles 
(1914g).  Escher  (1909)  was  unable  to  determine  the  constitution  of 
carrot  carotin  although  he  had  at  his  disposal  150  grams  of  pure 
pigment. 

Other  yellow  roots,  such  as  parsnip,  sweet  potato,  yellow  turnip, 
rutabaga,  squash,  etc.,  undoubtedly  contain  carotinoids  but  the  exact 
nature  of  the  pigments  has  not  been  determined. 

The  existence  of  yellow  pigments  in  chloroplastids  was  discovered 
by  Fremy  (1860),  but  the  first  definite  separation  from  green  pig- 
ment was  made  by  Stokes  (1864),  and  later  by  Kraus  (1872)  and 
Sorby  (1873)   and  others. 

The  first  crystals  of  yellow  plastid  pigment  were  observed  by  Fremy 
(1865)  and  later  by  Hartsen  (1873a),  Bougarel  (1877),  Borodin 
(1883)  and  Guignet  (1885).  It  remained  for  Arnaud  (1885),  how- 
ever, to  observe  the  identity  of  these  crystals  with  carrot  carotin, 
which  was  confirmed  by  chemical  analysis  through  the  work  of  Im- 
mendorff  (1889)  and  Willstatter  and  Mieg  (1907). 

The  plurality  of  the  yellow  chloroplastid  pigments  was  first  sug- 
gested by  Stokes  (1864a)  and  definitely  demonstrated  by  Borodin 
(1883).  The  correct  procedure  for  the  separations  of  these  pigments 
as  well  as  their  present  classification  as  carotinoids  was  developed  by 
Tswett   (1906  to  1911)   on  the  basis  of  the  observations  of  Kraus 


CAROTIN OWS  IN  THE  PHANEROGAMS  89 

(1872)  and  Monteverde  (1893),  as  well  as  on  his  own  physico-chemi- 
cal studies  of  the  leaf  pigments.  Tswett's  theories  regarding  the 
chemical  relation  between  the  carotin  and  xanthophyll  groups  of 
carotinoids  were  substantiated  by  Willstatter  and  Mieg  (1907),  who 
isolated  the  first  crystalline  xanthophyll  and  established  for  it  the 
formula  C^qH^qOz-  Tswett's  adsorption  method  of  analysis  of  the 
carotinoids  in  chloroplastids  indicates  the  existence  of  at  least  four 
yellow  xanthophylls  accompanying  carotin  in  the  leaf.  The  crystal- 
line xanthophyll  isolated  by  Willstatter  and  Mieg  is  probably  a  mix- 
ture of  two  or  more  of  these  xanthophylls.  The  author  proposes  a 
colloidal  theory  to  explain  the  adsorption  method  of  analysis  which 
reveals  the  several  xanthophyll  pigments. 

Xanthophylls  for  the  most  part  are  yellow  in  color,  but  Monteverde 
and  Lubimenko  (1913b)  have  discovered  a  red  xanthophyll  which  they 
call  rhodoxanthin. 

The  types  of  carotinoids  in  etiolated  plants  and  their  relative  pro- 
portions have  not  been  studied  since  the  advent  of  the  present  caro- 
tinoid  classification  and  the  development  of  methods  for  their  separa- 
tion. A  review  of  the  older  studies  indicates,  however,  that  carotin 
is  concerned  in  the  etiolated  color,  but  the  evidence  is  not  clear  as  to 
the  character  and  extent  of  the  xanthophyll  distribution. 

It  seems  certain  that  carotinoids  are  concerned  in  part  in  the  pig- 
mentation of  naturally  yellow  and  yellow  spotted  leaves.  The  types 
of  carotinoids  and  their  relative  proportions  have  not  been  determined 
by  modern  methods. 

The  important  questions  to  be  answered  regarding  the  yellow  chro- 
molipoids  concerned  in  autumn  colorations  are:  (1)  are  the  yellow 
autumn  pigments  merely  the  carotinoids  already  present  in  the  chloro- 
plastids, (2)  are  these  augmented  or  replaced  by  other  yellow  pig- 
ments closely  related  to  the  normal  carotinoids  but  still  capable  of 
being  differentiated  from  them,  (3)  are  the  yellow  autumn  pigments 
entirely  new  substances?  The  most  recent  study  of  these  questions 
by  Tswett  (1908c)  and  Miss  Goerrig  (1917)  shows  definitely  that  the 
yellow  colors  are  not  due  to  entirely  new  pigments.  It  has  not  been 
determined  with  certainty,  however,  whether  or  not  the  chloroplastid 
carotinoids  are  slightly  modified  during  the  necrobiosis  or  to  what 
extent  new  yellow  pigments  play  a  part  in  the  autumn  colorations. 
Tswett  has  concluded  that  the  yellow  colors  are  due  entirely  to  a 
mixture  of  slightly  modified  carotinoids,  which  he  calls  autumn  xan- 
thophylls, but  which  the   author  believes  should  better  have  been 


90  CAROTINOIDS  AND  RELATED  PIGMENTS 

named  autumn  carotins.  Green  as  well  as  autumn  leaves  also  con- 
tain, according  to  Tswett,  colorless  water-  and  alcohol-soluble  chromo- 
gens  which  form  golden  yellow  salts  with  acids  and  alkalies,  particu- 
larly the  latter,  and  which  readily  oxidize  to  a  brown  color.  These 
pigments  are  regarded  by  Tswett  as  playing  a  part  at  times  in  the 
necrobiotic  colorations,  and  the  postmortal  colors  are  held  to  be  due 
entirely  to  these  pigments.  Miss  Goerrig's  conclusions  oppose  those 
of  Tswett  in  indicating  that  the  yellow  autumn  colors  are  due  in  part 
to  the  normal  unchanged  carotinoids  of  the  chloroplastids,  diminished 
somewhat  in  quantity  in  comparison  with  the  mid-summer  green 
leaves.  Miss  Goerrig  believes,  however,  that  the  chief  role  is  played 
by  new  yellow  pigments  soluble  in  water. 

Autumn  and  winter  reddening  is  due  at  times  to  red  carotinoids. 
In  some  cases  the  red  xanthophyll,  rhodoxanthin,  is  involved,  e.g.,  in 
arbor  vitae.  In  other  cases  the  red  carotin,  lycopin,  is  involved,  e.g., 
certain  conifers,  under  tropical  conditions.  For  the  most  part,  how- 
ever, red  autumn  colors  are  due  to  anthocyanins. 

The  vast  majority  of  yellow  to  orange-red  flowers  owe  their  color 
to  chromoplastids  containing  carotinoids.  Very  little  is  known,  how- 
ever, regarding  the  character  and  distribution  of  the  individual  caro- 
tinoids among  these  flowers.  In  general,  floral  colors  of  a  primrose  or 
sulfur-yellow  color  are  produced  by  water-soluble  non-carotinoids 
which  are  flavones,  anthocyanins  or  related  pigments.  The  latter  are 
usually  present  in  solution  in  the  cell  sap  in  contrast  with  carotinoids 
which  are  present  in  plastids.  The  reader  is  referred  to  the  tables 
showing  the  flowers  whose  color  is  due  chiefly,  if  not  entirely,  to 
carotinoids. 

Carotinoids  are  undoubtedly  the  cause  of  the  color  of  many  yellow 
to  orange  colored  fruits.  The  reader  is  referred  to  the  text  for  the 
presentation  of  our  present  knowledge  of  this  subject.  Red  tomato 
fruits  are  characterized  by  a  red  carotinoid  called  lycopin,  which  is  a 
chemical  isomer  of  carotin,  differing  from  it  only  in  color  and  certain 
physical  properties.  These  relations  were  recognized  first  by  Millardet 
(1876)  and  definitely  established  by  Willstatter  and  Escher  (1910). 
A.  and  G.  de  Negri  (1879)  first  suggested  the  identity  of  the  water- 
melon pigment  with  the  red  tomato  pigment,  a  supposition  finally 
proved  by  Monteverde  and  Lubimenko  (1913b).  The  red  pepper  pig- 
ment is  also  probably  lycopin. 

The  arils  and  carpellary  tissue  of  a. number  of  seeds  are  also  char- 
acterized by  carotinoids,  carotin,  xanthophylls,  lycopin  and  rhodoxan- 


CAROTINOIDS  IN  THE  PHANEROGAMS  91 

thin  having  been  found  in  specific  cases,  which  are  enumerated  in  the 
text.  Carotinoids  are  also  found  in  certain  seeds  whose  carpellary 
tissue  is  less  highly  colored.  The  cereal  grains  also  contain  carotin 
and  xanthophylls.  The  pigment  of  yellow  maize  is  characterized  by 
a  large  proportion  of  xanthophyll  carotinoid. 


Chapter  III 

Carotinoids  in  the  Cryptogams 

The  non-flowering  forms  of  plant  life,  the  chromolipoids  of  which 
are  considered  in  the  present  chapter,  are  as  abundantly  characterized 
by  pigments  as  the  phanerogamous,  or  flowering  forms.  Indeed, 
among  the  algae,  which  will  be  first  considered,  the  more  important 
classes  derive  their  names,  at  least  their  common  designations,  from 
their  general  distinguishing  color.  The  same  is  true  in  a  few  cases 
for  fungi,  for  example,  the  rusts. 

The  information  available  regarding  the  character  and  distribution 
of  carotinoids  among  the  lower  forms  of  plants  is,  on  the  whole,  more 
abundant  than  might  be  supposed.  Speaking  first  for  the  algae,  it  is 
surprising  to  fihd  that  our  knowledge  is  practically  complete  for  cer- 
tain of  the  classes,  particularly  the  red  and  brown  sea-weeds.  On 
the  other  hand,  fragmentary  information  only  is  available  for  other 
classes  of  algae,  so  that  the  subject  of  the  carotinoids  among  the 
algae  is  by  no  means  as  yet  a  closed  book.  Some  of  the  algae  seem 
to  owe  their  characteristic  color,  at  least  in  part,  to  carotinoids.  This 
is  true  of  the  brown  sea-weeds  as  a  class  in  the  living  condition.  Cer- 
tain species  among  other  classes  apparently  owe  their  color  entirely 
to  carotinoid  pigments,  for  example,  the  so-called  blood  algae  Haemo- 
tococcus  'pluvialis,  one  of  the  Chlorophyceae,  but  this  phenomenon 
does  not  seem  to  be  the  general  rule. 

Carotinoid-like  colors  are  more  common  among  the  fungi  than 
among  the  algae,  but  the  colors  in  many  cases  appear  to  be  due  to 
other  pigments.  In  general,  it  may  probably  be  stated  with  some 
degree  of  assurance  that  carotinoids  are  not  so  common  among  the 
fungi  as  among  the  algae.  In  fact,  many  fungi  appear  to  be  entirely 
devoid  of  carotinoid  pigment,  while  practically  all  classes  of  algae 
appear  to  contain  pigments  of  this  type  to  some  extent,  or  at  least 
to  give  reactions  which  may  be  thus  interpreted. 

The  study  of  the  carotinoids  which  appear  to  be  regularly  pro- 
duced by  bacteria  of  certain  species  is  practically  an  unexplored  field. 
Practically  nothing  is  known  regarding  the  character  and  distribution 

92 


CAROTINOIDS  IN  THE  CRYPTOGAMS  93 

of  the  carotinoids  which  appear  to  be  produced  by  certain  of  these 
organisms.  Splendid  opportunities  for  research  exist  also  in  regard 
to  the  factors  governing  the  kinds  and  amount  of  the  pigment  pro- 
duced in  each  case.  Such  a  study  offers  some  fascinating  possibilities 
in  connection  with  the  discovery  of  the  true  function  of  the  carotinoids 
in  plants.  No  matter  how  acceptable  the  theories  appear  to  be  which 
are  at  present  in  the  ascendency  regarding  this  function,  it  is  to  be 
admitted  that  no  theories  have  been  advanced  which  can  claim  much 
experimental  basis.  It  seems  logical  to  assume  that  much  valuable 
information  might  be  secured  if  it  could  be  found  possible  to  control 
the  growth  and  character  of  carotinoids  in  simple  plants,  like  the 
bacteria.  If  the  carotinoids  are,  after  all,  merely  by-products  of  plant 
cell  activities  we  should  know  this  fact.  In  general,  as  plant  life 
ascends  the  scale  of  complexity  carotinoids  become  an  established 
product  of  the  cell  life,  and  their  invariable  appearance  in  the  chloro- 
plastids  has  been  interpreted  in  favor  of  a  functional  theory.  The 
fact  that  the  same  pigments  appear  at  times  in  other  organs  of  the 
chlorophyllous  plants  and  also  in  plants  which  lack  chlorophyll  en- 
tirely may,  however,  be  significant.  At  any  rate  the  possibility  is 
not  to  be  overlooked  of  throwing  some  light  on  this  question  through 
a  study  of  the  carotinoids  in  bacteria. 

Carotinoids  in  the  Algae 

The  plan  which  will  be  followed  will  be  to  present  the  available 
knowledge  regarding  each  class  of  algae  separately.  The  species  which 
have  been  examined  will  be  tabulated,  together  with  the  names  of  the 
investigators  and  the  dates  their  work  was  published.  The  author 
has  found  such  an  arrangement  helpful  in  the  study  of  the  subject 
and  believes  it  will  furnish  a  convenient  mode  of  reference  for  future 
workers  in  this  field.  The  order  of  presentation  of  the  various  classes 
follows  in  general  a  descending  scale  with  regard  to  complexity  of  the 
plant  forms. 

The  Phceophyceae.  These  plants,  commonly  known  as  the  brown 
or  olive-brown  sea-weeds,  comprise  a  large  group,  which  are  mostly 
marine  plants.  They  are  found  everywhere  in  the  seas,  especially  in 
the  colder  waters.  A  few  of  the  species  are  of  economic  importance. 
Laminaria  saccharina,  which  contains  the  carbohydrate  mannite,  is 
used  in  the  Orient  for  food.  The  carotinoids  of  this  class  of  algae  are 
better  known  both  qualitatively  and  quantitatively  than  the  chromo- 


9-1  CAROTINOIDS  AND  RELATED  PIGMENTS 

lipoids  of  any  other  class  of  algae.  The  fresh  plants  owe  their  olive- 
brown  tint  to  the  high  concentration  of  the  special  algae  carotinoid, 
fucoxanthin,  the  most  recent  member  of  the  chromolipoid  pigments 
to  be  brought  to  a  definite  chemical  conception.  The  dried  plants  owe 
their  brown  color  to  the  pigment  phycophain,  which  forms  in  the  plants 
after  death,  as  the  result  of  oxidation  of  colorless  chromogens  in  the 
cells.  The  belief  that  phycophain  is  the  characteristic  coloring  mat- 
ter of  the  brown  algae  still  finds  expression  in  the  text  books,  but  this 
is  only  true  of  the  dried  plants. 

The  brown  sea-weeds  which  have  been  examined  for  carotinoids  are 
given  in  Table  8.  The  table  shows  that  the  pigments  of  these  plants 
attracted  the  attention"  of  the  early  workers.  While  the  scope  of 
these  first  studies  was  naturally  limited  by  the  prevailing  knowledge 
of  the  plant  chromolipoids,  they  unquestionably  paved  the  way  for 
the  discovery  of  the  fucoxanthin  which  characterizes  the  brown  algae. 
The  fact  that  certain  species,  like  the  Fucoidiae  and  Laminaria,  are 
abundant  and  easily  obtained  no  doubt  accounts  for  their  popularity 
as  sources  of  material  for  investigation.  Of  the  various  investigations 
mentioned  in  the  table  those  of  Tswett  (1906),  Czapek  (1911),  Kylin 
(1912)  and  Willstatter  and  Page  (1914)  are  the  most  important.  The 
chief  contributions  of  these,  as  well  as  the  earlier  investigators,  to  the 
subject  may  be  summarized  as  follows: 

Rosanoff  (1867)  appears  to  have  first  expressed  the  belief  that  the 
Fucoidice  contain  a  special  pigment  besides  chlorophyll. 

Millardet  (1869),  working  in  Kraus'  laboratory,  submitted  the  abso- 
lute alcohol  extracts  from  several  species  of  both  the  dried  and  fresh 
plants  to  the  benzene  separation  method  which  had  just  been  worked 
out  by  Kraus.  The  yellow  pigment  remaining  in  the  alcohol  layer 
was  regarded  as  differing  from  the  xanthophyll  of  higher  plants  and 
was  called  phycoxanthin,  the  name  which  Kraus  and  Millardet  (1868) 
had  already  given  to  the  pigment  prepared  in  the  same  manner  from 
green  algae  and  diatoms.  Millardet  was  also  the  discoverer  of  the 
brown  water-soluble  pigment  of  the  PhceophycecB,  to  which  the  name 
phycophain  was  given.  Reinke  (1886)  first  expressed  the  belief  that 
phycophain  is  a  post-mortem  product.  Molisch  (1905)  first  offered 
experimental  proof  of  this  fact  which  was  later  definitely  proved  by 
Tswett  (1906)  and  confirmed  by  Kylin  (1912). 

Askenasy  (1869)  noticed  that  the  yellow  pigment  discovered  by 
Millardet  in  the  brown  algae  turns  blue  when  its  alcoholic  solution  is 
treated  with  HCl,  a  reaction  which  appears  to  be  specific  for  fuco- 


CAROTINOIDS  IN  THE  CRYPTOGAMS  95 

xanthin,  and  is  of  considerable  importance  for  the  qualitative  detec- 
tion of  this  pigment. 

Sorby  (1873)  proposed  the  name  fucoxanthin  for  the  chief  coloring 
matter  of  the  brown  algae.  He  differentiated  the  pigment  from  the 
xanthophyll  of  the  higher  plants  by  reason  of  the  position  of  its  spec- 
troscopic absorption  bands,  by  its  greater  resistance  to  the  bleaching 
action  of  light,  and  by  the  blue  color  obtained  when  HCl  is  added  to 
the  alcoholic  solution  of  the  pigment,  all  of  which  characteristic  prop- 
erties have  since  been  confirmed  for  the  pure  pigment.  Sorby  also  iso- 
lated "orange  xanthophyll"  (carotin)  from  brown  algae,  and  was  thus 
the  first  to  show  the  presence  of  the  better  known  carotinoids  in  these 
plants. 

Reinke  (1876)  proposed  the  name  phaophyll  for  the  special  yellow 
pigment  of  Phceophycece. 

Hansen  (1884d)  regarded  the  yellow  pigment  of  brown  algae  as 
identical  with  the  "yellow  chlorophyll"  of  higher  plants. 

Tammes  (1900)  demonstrated  the  presence  of  carotinoids  in  a  num- 
ber of  species  of  Phceophycece,  using  the  Molisch  alkali  microchemi- 
cal  crystallization  method.  It  is  now  known  that  he  was  mistaken 
in  attributing  the  result  to  carotin  only. 

Gaidukov  (1903),  however,  denied  that  either  carotin  or  a  special 
pigment,  phycoxanthin  (using  Millardet's  terminology),  characterizes 
the  brown  sea-weeds,  claiming  to  have  found  in  additibn  to  chlorophyll 
only  the  xanthophylls  which  characterize  the  higher  plants. 

Table  8.    Phaeophyceae  Found  to  Contain  Carotinoids 

Order  Cyclosporales  (highest  forms). 

Fucoidiae—RosanoE.  1867;  Czapek,  1911. 

Fucm  serratus—Mi\lg,vdet,  1869;  Sorby,  1873;  Reinke,  1876;  Tammes, 

1900;  Gaidukov,  1903;   Molisch,  1905;  Tswett,  1905,  1906;  Kylin, 

1912;  Willstatter  and  Page,  1914;  van  Wisselingh,  1915. 
Fucm    wescw/osr/^— Millardet,    1869;    Hansen,    1884;    Tammes,    1900; 

Tswett,  1906;  Kylin,  1912;  van  Wisselingh,  1915. 
Fucus  nodosus — Millardet,  1869. 
Fucits  versoides — Molisch,  1905. 
Ascophyllum  nodosum^Tammes,  1900;  Kylin,  1912;  van  Wisselingh, 

1915. 
Phyllospora  Brodiaei,  P.  membranifolia — Kylin,  1912. 
Cystoseira  abrotanifolia — Molisch,  1905. 
Halidrys   siloquosa—Millsirdet,    1869;    Reinke,    1876;    Molisch,    1905; 

Kylin,  1912. 
Order  Phceosporales. 

Ectocarpaceae — Askenasy,  1869. 

PylaieUa  litoralis — Kylin,  1912. 

Desmarestia  aculeata — Reinke,  1876. 

Elachista  species— Millardet,  1869;  Molisch,  1905. 


96  CAROTINOIDS  AND  RELATED  PIGMENTS 

Leathesia  manna— Millardet,  1869;  Molisch,  1905. 

Chorda  filum^T&mmes,  1900. 

Laminaria  sacc/iarma— Millardet,  1869;  Reinke,  1876;  Tammes,  1900; 

Molisch,  1905;  Tswett,  1905,  1906;  van  Wisselingh,  1915. 
Laminaria  digitalis— Tammes,  1900;  Molisch,  1905;  Kylin,  1912;  Will- 

statter  and  Page,  1914;  van  Wisselingh,  1915. 
Cutleria  multifida— Millardet,  1869;  Molisch,  1905. 
Orflcr  Dzct^otdiGS 

Dictyoia  dichotoma—Millavdet,  1869;  Molisch,  1905. 

Dictyopteris    polypodioides— Tammes,    1900;    Molisch,    1905;    Kylin, 

1912. 
Halyseris  polypodioides— Millardet,  1869;  Molisch,  1905. 
Padina  Pavonia — Molisch,  1905. 

Molisch's  (1905)  contributions  to  the  carotinoids  of  brown  algae 
were,  (1)  in  showing  that  the  water-soluble  phycophain  exists  only 
in  the  dried  plants  or  those  which  have  been  placed  in  hot  water  for 
a  few  minutes,  (2)  in  rediscovering  the  blue  color  reaction  with  HCl. 
Molisch  obtained  the  latter  reaction  either  by  extracting  the  fresh 
plants  with  alcoholic-HCl  (98  volumes  of  alcohol  and  2  volumes  of 
con.  HCl)  or  by  adding  HCl  to  the  alcoholic  layer  after  the  Kraus 
separation,  using  petroleum  ether  for  extracting  the  carotin  and 
chlorophyll.  Molisch,  however,  did  not  attribute  this  reaction  to  a 
carotinoid,  but  to  a  colorless  "leucocyan"  in  the  plant,  which  gave  rise 
to  a  blue  "phaeocyan"  with  HCl. 

Tswett  (1905)  was  quick  to  point  out  Molisch's  error  with  respect 
to  the  so-called  l§ucocyan  reaction,  showing  that  this  was  due  to  the 
special  carotinoid,  fucoxanthin,  in  the  plants,  as  Sorby  (1873)  had 
pointed  out  many  years  earlier.  Tswett  (1906)  was  thus  led  to  make 
a  closer  study  of  the  Phaeophyceae  pigments,  using  Fuctis  and  Lami- 
naria for  his  material.  He  showed  first  that  phycophain  is  a  post- 
mortem oxidation  product  and  does  not  exist  in  the  living  plants.  He 
next  made  a  careful  examination  of  the  chromatophor  pigments  of  the 
living  plants  making  use  of  the  relative  solubility  and  chromatographic 
adsorption  properties  of  the  carotinoids.  By  this  means  he  showed 
definitely  that  at  least  three  carotinoids  are  present,  namely,  fuco- 
xanthin, carotin,  and  xanthophyll.  The  former  is  the  principal  pig- 
ment. It  corresponds  to  the  xanthophylls  of  the  higher  plants  in  being 
adsorbed  from  pure  petroleum  ether  and  carbon  disulfide  by  calcium 
carbonate  and  other  finely  divided  materials,  and  by  remaining  hypo- 
phasic  in  the  Kraus  separation  between  petroleum  ether  and  80  per 
cent  alcohol.  It  differs  from  the  xanthophylls  of  the  higher  plants  in 
the  position  of  its  absorption  bands  in  the  spectrum,  by  the  reddish- 
brown  color  of  its  concentrated  solutions,  by  the  fact  that  it  is  attacked 


CAROTINOIDS  IN  THE  CRYPTOGAMS  97 

by  alkalies,  and  by  its  color  reaction  with  HCl.  Tswett  noticed,  like 
Sorby,  that  alkali  would  restore  the  yellow  color  of  alcoholic  solu- 
tions turned  blue  with  acid,  but  mentioned  that  the  shade  of  yellow 
was  not  quite  the  same  as  the  original.  Tswett  regarded  the  carotin 
present  as  identical  with  the  carotin  of  higher  plants.  The  xantho- 
phyll,  however,  was  probably  erroneously  regarded  as  a  special  xan- 
thophyll,  to  which  Tswett  gave  the  name  fucoxanthophyll.  Tswett 
also  studied  the  chlorophyllins  of  the  brown  algae,  finding  chloro- 
phyllin  a  (chlorophyll  a  of  Willstatter)  and  chlorophyllin  y,  a  spe- 
cial pigment,  which  he  regarded  as  characteristic  of  the  Phceophycece. 
This  has  not  been  confirmed  by  Willstatter  and  Page  (1914)  who 
found  only  chlorophyll  a  in  the  brown  algae. 

Czapek  (1911)  submitted  petroleum  ether  extracts  of  carefully 
dried  Fucoidece  to  a  Tswett  chromatographic  analysis  and  found 
chlorophyll  a,  fucoxanthin  and  xanthophyll,  but  no  carotin.  The  fail- 
ure to  find  carotin  was  probably  due  to  the  fact  that  Laminaria, 
according  to  Willstatter  and  Page,  contain  very  small  quantities  of 
carotin. 

Kylin  (1912)  has  given  us  one  of  the  best  systematic  examinations 
of  the  carotinoids  of  the  Phaeophyceae.  Although  Kylin  falls  into  the 
error  of  regarding  the  Molisch  microchemical  crystallization  test, 
which  he  performed  on  a  large  number  of  species,  as  specific  for  caro- 
tin, he  nevertheless  succeeded  in  isolating  the  first  crystals  of  this  pig- 
ment from  brown  algae.  Impure  crystals  of  xanthophyll  were  also 
secured.  An  unsuccessful  attempt  was  also  made  to  secure  crystals 
of  fucoxanthin,  for  which  pigment  Kylin  prefers  the  name  phycoxan- 
thin.  Kylin  pointed  out  the  probable  close  chemical  relation  of  fuco- 
xanthin to  xanthophylls.  He  found  that  a  greater  solubility  in  petro- 
leum ether  is  one  of  the  distinguishing  differences,  a  result  which 
Willstatter  and  Page  (1914)  find  is  characteristic  of  the  impure  pig- 
ment, but  not  of  the  pure  crystals.  The  latter  are  insoluble  in 
petroleum  ether.  Kylin  made  the  interesting  discovery  that  the  blue 
color  reaction  is  given  not  only  by  the  mineral  acids,  but  by  acetic 
and  oxalic  acids  as  well,  and  that  dilute  alkali  changes  the  pigment 
so  that  the  tendency  to  give  this  reaction  is  greatly  accelerated. 

Fucoxanthin.  The  .chemical  relation  of  fucoxanthin  to  the  other 
carotinoids  is  now  known  through  the  work  of  Willstatter  and  Page 
(1914),  who  also  determined  the  quantitative  distribution  of  the  dif- 
ferent carotinoids  in  the  olive-brown  sea-weeds.    Ultimate  analyses 


98 


CAROTINOIDS  AND  RELATED  PIGMENTS 


carried  out  on  five  different  preparations  of  fucoxanthin  gave  the  fol- 
lowing results  in  comparison  with  several  theoretical  values. 


Found 

Calculated  for 

040-115408 

1^40  xlsevJe 

O41JJ.50U6 

C  =  76.39 
H  =    8.77 

76.14 
8.63 

75  90 
8.93 

76.35 
8.76 

The  investigators  prefer  the  formula  C^oHsiOg  over  C^oHggOg, 
although  the  latter  expresses  the  closer  empirical  relation  to  carotin, 
but  admit,  at  the  same  time,  that  their  data  correspond  most  closely 
to  the  formula  containing  one  additional  carbon  atom.  In  any  case 
the  close  chemical  relation  of  fucoxanthin  to  the  other  carotinoids  is 
clearly  established. 

The  observations  of  Tswett  and  of  Willstatter  and  Page  show  that 
great  care  must  be  taken  in  the  isolation  of  fucoxanthin  to  prevent 
the  formation  of  the  post-mortal  phycophain.  This  was  prevented  by 
Willstatter  by  dehydrating  the  fresh  plants  with  30  per  cent  acetone, 
after  which  the  material  was  macerated  and  extracted  at  once  with 
pure  acetone.  The  extract  containing  the  combined  chlorophyll  and 
carotinoids  was  then  diluted  with  ether  which  was  washed  free  from 
acetone  with  water.  The  ether  extract  was  now  diluted  with  an  equal 
volume  of  low  boiling  petroleum  and  the  mixture  submitted  to  a  modi- 
fied Kraus  separation  using  70  per  cent  methyl  alcohol.  In  this  con- 
nection Willstatter  and  Page  made  the  valuable  observation  that 
fucoxanthin  is  quantitatively  removed  by  70  per  cent  alcohol  in  the 
Kraus  separation,  leaving  the  other  carotinoids  practically  quantita- 
tively in  the  petroleum  ether,  especially  if  ether  is  also  present.  It  is 
clear  that  this  discovery  not  only  permits  the  separation  of  fucoxan- 
thin but  does  not  interfere  with  the  subsequent  separation  of  xan- 
thophyll  and  carotin  by  the  usual  procedure  using  petroleum  ether 
and  80  per  cent  methyl  alcohol. 

Fucoxanthin  isolated  on  the  above  principle  was  found  to  crystal- 
lize readily  from  methyl  alcohol  or  acetone  in  dark  red  regular 
hexagons,  containing  water  or  alcohol  of  crystallization,  the  latter 
being  lost  only  in  high  vacuum  at  105°  C.  By  precipitating  the  pig- 
ment from  ether. with  low  boiling  petroleum,  in  which  it  is  insoluble, 
compact  needles  without  any  solvent  of  crystallization  were  obtained. 

A  study  of  the  characteristic  co^or  reaction  with  HCl  showed  it  to  be 


cArotinoids  in  the  cryptogams  99 

due  to  the  formation  of  a  hydrochloride  of  the  composition  C^^oRr,/)^, 
4HC1,  the  yellow  pigment  restored  by  alkali  still  containing  one  mole- 
cule of  HCl.  The  instability  of  the  pigment  in  the  presence  of  strong 
alkali,  which  was  observed  by  Tswett,  was  confirmed  on  the  pure 
substance,  it  being  shown  that  a  new  substance  was  formed  which, 
when  set  free  from  the  alkali  by  water,  showed  characteristic  spectro- 
scopic and  solubility  properties.  It  was  found  that  the  effect  of  alkali 
in  increasing  the  sensitiveness  of  fucoxanthin  solutions  towards  the 
blue  color  reaction  with  acids,  which  Kylin  noted,  was  due  to  this 
modified  pigment.  Willstatter  and  Page  observed  that  as  little  as 
0.001  per  cent  HCl  would  give  the  blue  color  with  a  concentrated 
ether  solution  of  the  pigment,  after  its  modification  with  alkali. 

Fucoxanthin  appears  to  be  an  even  more  intense  pigment  than 
carotin  or  xanthophyll  from  the  observations  of  Willstatter  and  Page, 
who  found  that  85  mm.  of  0.2  per  cent  KgCrsO^  has  the  color  equiva- 
lent of  108  mm.  of  xanthophyll,  or  80  mm.  of  carotin,  but  only  50  mm. 
of  fucoxanthin,  using  in  each  case  a  5  X  10"^  molar  concentration 
of  pigment. 

Other  interesting  properties  of  fucoxanthin  observed  by  these  inves- 
tigators were  the  formation  of  oxonium  salts,  a  crystalline  iodide, 
C^oHg^Ogl^,  and  a  bleached  oxidation  product  C^qH^JJj^q.  In  con- 
nection with  the  last  named  product  it  was  found  that  the  crystals  of 
pigment  are  much  more  stable  than  carotin  or  xanthophyll,  but  that 
the  solutions,  especially  benzene  solutions,  bleach  readily. 

Regarding  the  quantitative  distribution  of  the  carotinoids  in  Phaeo- 
phyceae,  Willstatter  and  Page  give  the  following  figures  on  both  the 
fresh  and  dry  basis  for  species  representing  the  three  principal  orders 
of  these  plants. 

Fresh  Algae  Dry  Algse 

Fucoxan-  Xantho-  Fucoxan-  Xantho- 

thin  Carotin  phyll  thin  Carotin  phyll 

per  cent  per  cent  per  cent  per  cent  per  cent  per  cent 

Fucus             0.0169  0.0089  0.0087  0.0593  0.0312  0.0305 

Dictyota          .0250  .0057  .0063             

Laminaria       .0081  .0006  .0038  .0528  .0038  .0243 

Willstatter  and  Page  gave  some  attention  to  the  character  of  the 
xanthophyll  present.  They  were  unable  to  observe  any  properties 
which  would  serve  to  distinguish  the  xanthophyll  isolated  by  them 
from  the  crystalline  xanthophyll  of  higher  green  plants.  This  result 
throws  doubt  on  the  existence  of  a  special  fucoxanthophyll  in  the 


100  CAROTINOWS  AND  RELATED  PIGMENTS 

brown  algae,  as  Tswett  concluded.  The  question  still  remains  open, 
however,  as  to  whether  more  than  one  xanthophyil  is  present. 

The  Rhodophyceae.  These  plants,  commonly  known  as  the  red  sea- 
weeds, are,  like  the  brown  algae,  found  mostly  in  salt  waters,  only  a 
few  inhabiting  fresh  water.  Both  forms,  fortunately,  have  been  ex- 
amined for  carotinoids.  The  plants  are  especially  abundant  in  the 
tropic  oceans  and  in  the  temperate  regions  at  lower  depths.  Several 
hundred  species  have  been  described.  The  dried  thallus  of  Chondrus 
crispus  forms  the  carragheen,  or  dried  moss  which  is  used  for  its  gela- 
tion properties.  Various  species  of  these  plants  are  the  source  of  agar- 
agar.  The  thallus  of  the  Rhodofhyceae  is  abundant  in  pigment  and 
may  be  red,  violet  or  purple,  but  rarely  green.  The  characteristic 
pigment,  however,  never  appears  to  be  carotinoid.  Some  chlorophyll 
appears  to  be  present,  but  the  chief  pigment  is  a  protein-like  material 
or  is  combined  with  such  (Kylin,  1911),  and  is  known  as  phyco- 
erythrin. 

The  red  algae,  however,  do  not  lack  carotinoids.  Those  in  which 
the  chromolipoids  have  been  demonstrated  are  given  in  Table  9.  The 
investigations  upon  which  our  knowledge  of  the  carotinoids  in  the  red 
algse  depends  may  be  summarized  as  follows. 

Sorby  (1873)  appears  to  have  first  called  attention  to  the  presence 
of  yellow  pigments  in  this  class  of  plants,  when  he  was  able  to  demon- 
strate the  presence  of  xanthophyil  in  Porphyra  vulgaris.  It  will  be 
recalled  from  the  summary  of  Sorby's  work  given  in  Chapter  II  that 
his  "xanthophyil"  corresponds  in  properties  with  the  xanthophyil  a 
of  the  higher  plants  as  revealed  by  Tswett's  chromatographic  analysis, 
and  also  to  the  chief  properties  of  the  crystalline  xanthophyil  isolated 
from  green  plants  in  Willstatter's  laboratory. 

Reinke  (1876)  extracted  Batrachospermum  moniliforme  with  hot 
alcohol,  and  obtained  a  yellow  extract  which  gave  up  its  pigment  to 
benzene.  This  result  indicates  carotin  in  the  light  of  our  present 
knowledge  of  the  relative  solubility  properties  of  the  carotinoids. 

Nebelung  (1878)  examined  the  effect  of  alcohol  and  petroleum  ether 
as  solvents  for  the  pigments  of  several  fresh  water  Rhodophyceae,  and 
found  that  a  yellow  pigment  (or  pigments)  could  be  extracted. 

Hansen  (1893)  applied  his  method  of  separating  green  and  yellow 
pigments  to  the  alcoholic  extracts  of  a  number  of  red  algae.  He 
found  that  green  and  yellow  fractions  could  be  obtained  by  treating 
the  extracts  with  alkali  and  shaking  with  ether.     He  regarded  the 


CAROTINOIDS  IN  THE  CRYPTOGAMS  101 

result  as  indicative  of  the  same  types  of  pigments  as  are  present  in 
higher  plants  which  he  had  studied  with  like  results. 

Tammes  (1900)  demonstrated  carotinoids  in  three  species  of  red 
algae,  which  are  mentioned  in  Table  9,  using  the  Molisch  micro- 
crystallization  method.  Kohl  (1902),  using  the  same  method,  con- 
firmed this  work  as  well  as  obtaining  positive  results  on  other  species. 

Table  9.    Rhodophyceae  Found  to  Contain  Carotinoids 

Order   Bangiales 

Bangia  species  (fresh  water) — Nebelung,  1878;  Kohl,  1902. 

Porphyra  laciniata — Tammes,  1900. 

Porphyra  hiemalis — Kylin,  1911. 

Porphyra  vulgaris — Sorby,  1873. 
Order   Nemalionales 

Lemania  fluviatilus  (fresh  water) — Nebelung,  1878;  Kohl,  1902. 

Batrachospermum   monilijorme    (fresh  water) — Reinke,   1876;    Nebelung, 
1878;  Kohl,  1902. 

Chantransia  species  (fresh  water) — Nebelung,  1878;  Kohl,  1902. 
Order   Gigartinales 

Chondrus  crisptis — Kylin,  1911;  van  Wisselingh,  1915. 

Cystoclonium  purpurascens — Kylin,  1911. 
Order   Rhodymeniales 

Dellesseria  sanguinea — Kylin,  1911. 

Laurencia  pinnatifida — Kylin,  1911. 

Polysiphonia  species — Tammes,  1900. 

Polysiphonia  nigrescens — Kylin,  1911. 

Rhodomela  subfusca,  R.  virgata — Kylin,  1911. 

Ceramium  rubrum — Tammes,  1900;  Kylin,  1911;  van  Wisselingh,  1915. 

Ceramium  diaphanum — Kylin,  1911. 

Callithamnion  hiemale — Kylin,  1911. 

Spermothamnion  roseolum — Kylin,  1911. 
Order   Cryptonemiales 

Dumontia  filijormis — Kylin,  1911. 

Furcellaria  fastigiata — Kylin,  1911. 

Polyides  rotundus — Kylin,  1911. 

Corallina  officinalis — Kylin,  1911. 

Kylin  (1911)  has  given  us  the  most  complete  study  of  the  carotin- 
oids of  the  Rhodophyceae.  He  first  successfully  applied  the  Molisch 
carotinoid  test  to  some  18  different  species  of  red  algse,  as  noted  in 
Table  9.  Unfortunately  this  test  is  not  specific  for  carotin  as  Kylin 
believed.  Ceramium  rubrum  was  employed  for  a  special  study  of  the 
carotinoids.  Both  carotin  and  xanthophylls  were  demonstrated  by 
applying  the  Kraus  procedure  to  extracts  of  the  plants.  There  seems 
to  be  no  question  regarding  the  presence  of  carotin  in  the  red  algse. 
The  study  of  the  xanthophylls  led  to  less  satisfactory  results.  By 
evaporating  the  xanthophyll-containing  alcohol  fraction  to  dryness 
and  heating  the  residue  with  petroleum  ether  it  was  found  possible 
to  separate  the  pigment  into  two  fractions.    The  fraction  which  dis- 


102  CAROTINOIDS  AND  RELATED  PIGMENTS 

solved  was  readily  re-extracted  from  the  solution  by  80  per  cent 
alcohol,  and  its  solubility  in  petroleum  ether  was  the  only  point  of 
difference  observed  between  the  two  xanthophylls.  Spectroscopic  and 
adsorption  properties  were  not  examined.  Both  xanthophylls  turned 
green  and  then  blue  on  addition  of  acids  to  their  alcoholic  solutions 
and  alkali  restored  the  yellow  color.  This  property  is  characteristic 
of  Tswett's  xanthophyll  (3  of  higher  plants,  as  pointed  out  in  the  pre- 
ceding chapter.  The  reaction  also  resembles  the  color  reaction  of 
fucoxanthin  with  acids.  Kylin,  himself  (1912),  suggests  this  in  a  foot- 
note of  the  report  of  his  study  of  the  pigments  of  the  brown  algae,  but 
was  unable  to  decide  whether  the  color  reaction  was  due  to  traces  of 
brown  algae  and  diatoms  present  with  his  material,  or  to  the  actual 
presence  of  fucoxanthin  in  the  red  algae.  It  is  doubtful  whether 
Kylin  really  effected  a  separation  of  distinct  pigments  in  his  xantho- 
phyll fractions.  It  is  not  at  all  unlikely  that  the  Rhodophycece  con- 
tain some  fucoxanthin.  A  spectroscopic  and  chromatographic  analysis 
of  the  xanthophylls  of  the  red  algae  as  well  as  an  application  of  the 
modified  Kraus  procedure  for  separating  fucoxanthin  from  the  other 
carotinoids  would  be  helpful  in  deciding  whether  the  color  reaction 
observed  by  Kylin  was  due  to  fucoxanthin  or  to  a  xanthophyll  of  the 
P  type. 

Van  Wisselingh  (1915)  has  recently  demonstrated  carotinoids  in 
two  species  of  red  algae  using  both  the  Molisch  and  the  acid  micro- 
crystallization  method. 

The  Charales.  This  class  of  algae,  commonly  known  as  the  stone- 
worts,  have  the  interesting  property  of  depositing  calcium  from  the 
waters  in  which  they  thrive,  from  which  they  derive  their  popular 
name.  Only  two  genera  are  known,  namely,  Chara  and  Nitella.  Both 
Tammes  (1900)  and  Kohl  (1902)  were  successful  in  showing  caro- 
tinoids to  be  present  in  Chara  fragilis,  using  the  Molisch  method. 
The  same  result  was  obtained  by  van  Wisselingh  (1915)  on  Nitella 
spores.  The  evidence  points  to  the  presence  of  carotinoids  in  the 
stoneworts,  but  nothing  further  is  known  regarding  their  character. 

The  Chlorophyceae.  The  so-called  green  algae  constitute  one  of  the 
largest  and  most  important  classes  of  lower  plants.  They  are  found 
in  both  fresh  and  salt  water,  but  the  former  predominate.  The  cells 
as  a  rule  contain  chloroplastids  which  makes  the  question  of  the  types 
of  plastid  pigments  present  an  important  one.  The  feature  which 
has  especially  attracted  attention,  however,  is  the  fact  that  the  spores 


CAROTINOIDS  IN  THE  CRYPTOGAMS  103 

of  certain  species,  e.g.,  Haematococcus,  in  the  resting  stage  are  charac- 
terized by  a  deep  red  or  violet  pigmentation.  The  species  which  have 
been  examined  for  carotinoids  represent  all  the  important  orders. 
These  are  given  in  Table  10. 

The  development  of  the  question  of  the  character  of  the  pigments 
other  than  chlorophyll  which  characterize  the  green  algae  has  followed 
closely  the  development  of  the  ideas  regarding  the  carotinoids  in  the 
higher  plants,  as  given  in  Chapter  II.  For  example,  the  earliest 
workers,  i.e.,  Cohn  (1850),  DeBary  (1856),  Caspary  (1858),  Hilde- 
brand  (1861)  and  Frank  (1877)  observed  merely  that  the  yellow  or 
red  pigments  present  in  the  algae  which  they  examined  responded  to 
extraction  by  the  fat  solvents  and  gave  .either  the  blue  color  reaction 
with  concentrated  HgSO^,  characteristic  of  the  pigments  later  known 
as  lipochromes,  or  the  blue  color  with  iodine  which  also  characterizes 
these  pigments.  The  spectroscopic  studies  of  Nebelung  (1878)  unfor- 
tunately contributed  very  little  to  the  elucidation  of  the  character  of 
the  pigments  present.  Klebs  (1881)  made  a  more  thorough  study 
of  the  properties  of  the  "yellow  oil"  in  certain  species,  and  mentions 
properties  now  well  recognized  as  class  characteristics  of  the 
carotinoids. 

Borodin  (1883)  appears  to  have  first  definitely  recognized  the  rela- 
tion of  the  yellow  pigments  of  the  Chlorophyceae  to  those  of  higher 
plants  and  isolated  red  rhombic  carotin  crystals  from  Spirogyra. 
Molisch  (1896),  Tammes  (1900)  and  Kohl  (1902)  as  well  as  van 
Wisselingh  (1915)  have  also  demonstrated  the  presence  of  carotinoids 
in  a  number  of  species  using  the  Molisch  micro-crystallization  test. 

Rostafinski  (1881)  first  expressed  the  possibility  of  xanthin  (older 
terminology  for  carotin)  being  present  in  the  species  of  green 
algae  which  he  examined.  Willstatter  and  Page  have  not  only  shown 
that  this  is  the  case  for  Ulva  lactuca  but  have  determined  both  carotin 
and  xanthophyll  quantitatively  in  this  species.  The  amounts  found 
were  0.0243  grams  of  carotin  and  0.0643  grams  of  xanthophyll  per 
kilo  of  fresh  material.  The  interesting  point  here  is  that  practically 
the  same  proportion  between  carotin  and  xanthophyll  is  found  to 
exist  in  this  species  of  algae  as  in  the  leaves  of  the  flowering  plants. 

Attention  has  already  been  called  to  the  interesting  phenomenon 
of  the  red  color  of  certain  species  of  Chlorophyceae  in  the  resting 
stage.  Special  study  of  the  character  of  the  pigments  present  in 
Sphaerelln     {Haematococcus  =  Chlamydococcus)    pluvialis,    the    so- 


104  CAROTINOIDS  AND  RELATED  PIGMENTS 

called  blood  algee,  and  Trentepohlia  Jolithus,  the  so-called  violet 
algae,  has  been  made  by  Rostafinski  (1881),  Karsten  (1891)  and 
Zopf  (1892a,  1895).  Cohn  (1850)  first  called  the  pigment  haemato- 
chrome  and  this  name  was  adopted  by  a  number  of  subsequent  inves- 
tigators for  the  red  pigment  of  many  species  of  green  algae.  Rosta- 
finski attempted  to  associate  the  haematochrome  with  chrysoquinone, 
because  the  latter,  like  the  red  algae  pigment,  gave  the  blue  color 
reaction  with  concentrated  H2SO4.  A  spectroscopic  comparison  of  the 
two  pigments  failing  to  substantiate  such  an  identity  the  name  chloro- 
rufin  was  proposed  for  the  algae  pigment,  and  the  possibility  expressed 
of  an  identity  with  the  solanorubin  (lycopin)  which  had  been  de- 
scribed a  few  years  previously  by  Millardet  (1876).  Karsten  observed 
that  the  haematochrome  extracted  from  Trentepohlia  by  absolute 
alcohol  stained  brown  with  osmic  acid,  a  reaction,  however,  which 
may  have  been  due  to  traces  of  fat  present  in  the  extracts. 

Table  10.    Chlorophyceae  Found  to  Contain  Carotinoids 

Order    Oedogoniales  (highest  forms). 

Oedogonium  species — Tammes,  1900;  Kohl,  1902;  van  Wisselingh,  1915. 

Bulhochaete—DeBaxy,  1856;  Cohn,  1867. 

Bulbochaete  setigera — Kohl,  1902, 
Order   Heterosiphonales 

Botrydium-^RostSi&nski,  1881. 
Order  Conjugatce 

Spirogyra  crassa — Borodin,   1883;    Molisch,   1896;   Tammes,   1900;    Kohl, 
1902. 

Spirogyra  maxima — Borodin,  1883;  van  Wisselingh,  1915. 

Zygnema  cruciatum — van  Wisselingh,  1915. 
Order    Ulotrichales 

Phycopeltis  epiphyton.  P.  Teuhii,  P.  maritima,  P.  aurea,  P.  amboinemis — 
Karsten,  1891;  Zopf,  1892a. 

Cephaleurus  (Mycoidea)  leavis,  C.  solutus,  C.  albidus,  C.  parasiticus,  P. 
minimus — Karsten,  1891;  Zopf,  1892a. 

Trentepohlia    (Chroolepv^)    maxima,   T.  m,onilijormis,   T.  crassiaepta,   T. 
bisporangiata,  T.  cyania — Karsten,  1891 ;  Zopf,  1892a. 

Trentepohlia  jolithus— Karsten,  1891;  Zopf,  1892a;  Kohl,  1902. 

Trentepohli-a  aurea — Rostafinski,  1881 ;  Zopf,  1892a. 

Trentepohlia  umbrina — Caspary,  1858;  Frank,  1877;  Zopf,  1892a. 

Trentepohlia  aureum-tomentosum — Caspary,  1858;  Hildebrand,  1861. 

Trentepohlia—Cohn,  1867. 

Stichococcus  majus — van  Wisselingh,  1915. 
Order    Ulvales 

Ulva  lactuca  (Sea  lettuce) — Willstatter  and  Page,  1914. 

Enteromorpha  intestinalis — Tammes,  1900. 
Order   Siphonocladiales 

Cladophora  glomerata — Nebelung,  1878;  Tammes,  1900;  Kohl,  1902;  van 
Wisselingh,  1915. 

Sphaeropleacea — Cohn,  1867. 
Order   Siphonales 

Vaucheria  species — Nebelung,  1878. 

Chlorella  protothecoides,  C.  variegata — van  Wisselingh,  1915. 


i 


CAROTINOIDS  IN  THE  CRYPTOGAMS  105 

Order  Protococcales  (lowest  forms,  unicellular) 

Sphaerella    (Haematococcus   or   Chlamydococcus)    pluvialis — Cohn,    1850, 

Eostafinski,   1881;    Klebs,   1883;    Zopf,   1895;    Kohl,    1902;    Jacobsen, 

1913;  van  Wisselingh,  1915. 
Volvox— Cohn,  1867. 

Protococcus  {Pleurococcus)  pluvialis — Cohn,  1850,  1867. 
ProtococciLs  vulgaris — van  Wisselingh,  1915. 
Scotinosphaera  paradoxa — Klebs,  1881. 
Phyllohium  dimorphum,  P.  incertums — Klebs,  1881. 
Hydrodictyon  utriculatum  (water-net) — Tammes,  1900. 

In  Zopf's  (1892a)  first  study  of  haematochrome  it  was  pointed  out 
that  the  previous  investigations  of  the  pigment  were  made  on  impure 
mixtures.  Zopf  not  only  succeeded  in  isolating  practically  pure  crys- 
tals of  the  pigment  from  Trentepohlia  Jolithus,  but  also  established 
their  identity  in  form  and  properties  with  the  carotin  from  carrots. 
Zopf  later  (1895)  was  led  to  compare  the  blood  and  violet  algae  from 
a  pigment  standpoint.  It  was  found  that  the  fresh  vegetation  of  the 
latter  presents  a  brighter  red  appearance,  the  cells  under  the  micro- 
scope appearing  yellow  to  orange;  Sphaerella  pluvialis,  on  the  other 
hand,  appears  dark  red-brown  when  fresh,  even  thin  layers  in  water 
having  a  blood  red  color,  the  same  coloration  appearing  under  the 
microscope.  These  and  other  differences  led  to  a  special  examination 
of  what  appeared  to  be  a  special  pigment  in  Sphaerella  (Haematococ- 
cus) pluvialis  different  from  the  carotin  in  the  Trentepohlia.  Two 
carotinoids  were  found,  namely,  carotin  proper,  which  Zopf  called 
"eucarotin,"  and  a  red  carotinoid-like  pigment  which  is  called  "caro- 
tinin,"  the  latter  being  the  predominating  pigment. 

Special  interest  attaches  to  this  red  "carotinin."  Its  properties,  as 
described  by  Zopf,  are  characterized  by  combining  readily  with  alka- 
lies and  by  showing  only  one  wide  spectroscopic  absorption  band  at 
the  F  line.  In  other  respects  it  has  the  class  characteristics  of  a 
carotinoid.  Some  doubt,  however,  is  thrown  upon  the  alleged  alkali 
combinations  of  the  red  "carotinin"  described  by  Zopf  by  the  obser- 
vations of  van  Wisselingh  (1915),  who  made  a  special  study  of  the 
response  of  the  blood  algae  to  microchemical  crystallization  tests,  as 
well  as  the  effect  of  various  reagents  on  the  pigment  crystals.  Three 
carotinoids  were  found.  The  Molisch  alkali  method  applied  to  the 
green  spores  of  the  algae  gave  red  platelets  insoluble  in  phenol- 
glycerin  (a  xanthophyll  solvent)  and  orange  needles  soluble  in  phenol- 
glycerin,  indicating  the  presence  of  carotin  and  xanthophyll.  The 
red  spores  when  treated  with  the  Molisch  reagent  gave  red-violet 
crystal   aggregates   which   contained  two   pigments,   one   an   orange 


106    .       CAROT^INOIDS  AND  RELATED  PIGMENT^ 

yellow  and  the  other  a  violet  colored  substance.  The  latter  was 
regarded  as  Zopf's  red  carotinin.  Its  xanthophyll  nature  was  shown 
by  its  ready  solubility  in  the  phenol-glycerin  reagent.  The  possibility 
of  this  pigment  being  a  potassium  compound  as  it  should  be  from 
Zopf's  description,  inasmuch  as  it  was  produced  in  a  strongly  alka- 
line medium  (Molisch's  reagent),  was  tested  by  treating  the  crystals 
with  dilute  acid  for  24  hours.  No  change  was  produced  in  their 
properties. 

The  red  pigment  of  the  blood  algae  deserves  further  study  in  the 
light  of  the  apparently  conflicting  observations  of  Zopf  and  van 
Wisselingh.  Its  red  color  and  xanthophyll-like  properties  as  de- 
scribed by  the  latter  investigator  suggests  the  red  rhodoxanthin  de- 
scribed in  Chapter  II.  No  other  carotinoids  haive  yet  been  described, 
however,  which  show  acid  properties  and  combine  with  alkalies  as  the 
red  algae  pigment  is  stated  to  do. 

An  interesting  observation  made  by  van  Wisselingh  in  connection 
with  his  study  of  the  blood  algae  was  that  the  plants  had  mostly  green 
aplanospores  when  cultivated  in  media  containing  0.01  per  cent  each 
of  KNO3,  (NHJ2HPO4,  MgCla,  Na^SO^,  but  that  the  aplanospores 
were  mostly  red  when  allowed  to  develop  in  media  containing  0.02  per 
cent  NH^NOg,  K2HPO4  and  MgSO^.  Jacobsen  (1913)  has  also  studied 
the  conditions  governing  the  formation  of  pigment  in  Haemato coccus 
pluvialis  and  found  that  temperature  as  well  as  food  conditions  influ- 
ence it.  He  was  unable  to  extract  the  pigment  from  the  plant  with 
fatty  oils,  and  it  did  not  respond  to  Tswett's  resorcin  method  for  the 
microcrystallization  of  carotinoid. 

The  Bacillariea  (Diatomaceae) .  The  diatoms  are  unicellular 
algae  of  very  peculiar  structure  and  interesting  habits.  The  single 
cells  are  composed  of  two  symmetrical  valves  which  are  held  together 
by  a  membranous  sac  of  slightly  colored  protoplasm.  The  single 
cells  are  10|x  or  less  in  diameter.  The  valves  of  which  they  are  con- 
structed are  frequently  beautifully  sculptured,  and  when  many  of  the 
cells  unite,  as  is  sometimes  the  case,  very  peculiar  shaped  structures 
often  result.  The  epidermis  of  the  diatoms  is  composed  of  silica  which 
these  organisms  have  the  power  to  extract  from  the  water  in  which 
they  develop.  Deposits  of  silica  from  great  growths  of  these  plants 
have  considerable  commercial  value  as  diatomaceous  earth.  The 
algae  inhabit  stagnant  water,  wet  rocks  and  the  sea. 

The  diatoms  comprise  a  considerable  portion  of  the  plankton  of  the 
sea.    It  is  this  fact,  together  with  the  part  which  the  plankton  of  the 


CAROTINOIDS  IN  THE  CRYPTOGAMS  107 

sea  plays  in  the  food  of  marine  animals  which  makes  the  pigments 
of  the  diatoms  of  interest.  Most  species  of  diatoms  have  a  brownish 
color.  A  few  are  green  and  probably  contain  chlorophyll,  or  at  least 
one  of  the  chlorophyll  pigments.  Of  the  many  thousands  of  species 
which  are  known,  unfortunately  only  a  few  have  been  examined  for 
carotinoids.  However,  these  are  probably  to  be  considered  as  typical 
of  the  remainder. 

The  earliest  workers  regarded  the  color  of  the  diatoms  as  due  to  a 
single  pigment  to  which  Nageli  (1849)  gave  the  name  diatomin.  This 
name  was  adopted  by  Askenasy  (1867)  for  the  brownish  yellow  pig- 
ment which  could  be  extracted  with  alcohol,  and  which  he  described 
as  showing  a  strong  absorption  of  the  blue  half  of  the  spectrum  and- 
a  characteristic  intense  blue-green  color  on  addition  of  H2SO4  or  HCl 
to  the  alcoholic  solution.  Nebelung  (1878)  extracted  a  yellow  pig- 
ment from  Melosira  species  with  petroleum  ether  but  called  it  phyco- 
xanthin.  The  principal  pigment  extracted  in  this  case  may  have  been 
carotin. 

Further  proof  of  the  carotinoid  nature  of  the  Bacillariea  pigments 
was  furnished  by  Tammes  (1900),  who  obtained  the  Molisch  test  on 
Fragilaria  species  and  by  Kohl  (1902)  who  obtained  the  same  test  on 
Gomphonema  and  Navicula  species.  Molisch  (1905),  himself  apply- 
ing the  test  to  Nitzschia  Palea,  Nitzschia  sigmoidea,  Cymatopleura 
solea  and  Pinnularia  viridis  {Navicula  viridis) ,  obtained  only  yellow 
drops,  but  these  gave  the  chromolipoid  color  reactions  with  H2SO4 
and  iodine. 

The  conclusions  of  the  various  investigators  are  somewhat  conflict- 
ing regarding  the  exact  nature  of  the  carotinoids  present  in  the  silice- 
ous algae.  Zopf  (1900)  concluded  that  "eucarotin"  (true  carotin) 
is  the  chief  pigment  present  in  Gomphonema,  but  that  the  pigment 
differs  somewhat  from  the  carotin  of  other  plants.  Kohl  (1906a) 
concluded  that  the  liver-colored  diatoms,  Achnanthidium  lanceolatum 
and  Eunotia  (Himanthidium)  pectinalis,  owe  their  color  chiefly  to 
carotin  with  a  little  of  his  so-called  (3-xanthophyll  (which  is  not 
carotinoid  in  the  true  sense)  present  also,  as  well  as  traces  of  chloro- 
phyll. Kohl  had  previously  (1902)  concluded  that  the  pigment  known 
as  diatomin  is  carotin.  Especially  interesting  is  the  observation  of 
Molisch  that  the  species  which  he  examined  (1905)  gave  the  so-called 
leucocyan  reaction  which  is  apparently  specifi.c  for  fucoxanthin. 
Askenasy  (1867)  had  observed  the  same  reaction  for  alcoholic  ex- 
.  tracts  of  diatoms,  so  that  there  are  at  least  strong  indications  that 


108  CAROTINOIDS  AND  RELATED  PIGMENTS 

fucoxanthin  is  present  in  these  algae.  The  view  of  Kohl  (1906)  that 
the  leucocyan  reaction  is  specific  for  carotin  is  hardly  to  be  regarded 
as  tenable.  The  imperfect  studies  which  have  been  made  do  not 
indicate  whether  true  xanthophylls  are  also  included  among  the  caro- 
tinoids  of  the  Bacillariea,  but  it  is  not  unlikely  that  this  will  be 
found  to  be  the  case  when  the  matter  comes  to  be  examined  in  detail. 

The  Peridinieae.  The  Peridiniales,  also  called  the  Dinoflagellata 
comprise  a  relatively  small  class  of  unicellular  algae,  which  are  found 
mostly  in  salt  (sea)  water.  They  sometimes  form  an  important  part 
of  the  plankton  of  the  sea,  so  that  their  pigments  are  of  interest,  as 
in  the  case  of  the  diatoms,  on  account  of  the  part  which  the  plankton 
of  the  sea  plays  in  the  food  of  fishes  and  other  marine  animals. 

Schiitt  (1890)  appears  to  have  made  the  only  specific  examination 
of  the  pigments  of  the  Peridinieae,  but  since  his  work  was  performed 
before  the  most  important  developments  took  place  in  the  field  of 
carotinoids  it  is  necessary  to  interpret  his  observations  in  the  light 
of  present-day  knowledge  of  the  subject.  Up  to  his  time  the  color 
of  the  Dinoflagellates  was  regarded  as  due  to  the  same  pigment  which 
was  believed  to  color  the  diatoms,  namely,  diatomin.  As  is  now 
known,  diatomin  is  not  a  specific  pigment.  Although  Schiitt  did  not 
recognized  this  fact  he  did  point  out  that  the  color  of  the  Peridinieae 
is  more  reddish-brown  and  easily  distinguished  from  the  yellowish- 
brown  color  of  the  diatoms.  This  difference  in  tint  was  found  to  be 
due  to  the  presence  of  carmine  colored  drops  or  globules  in  many  of 
the  Peridiniece  examined,  in  addition  to  the  yellowish-brown  pig- 
ment in  the  chromatophors  of  the  algae. 

The  Peridiniece  examined  by  Schiitt  were  Gymnodinium  Helix., 
Dinophysis  acuta,  D.  laevis,  Certium  tripos,  C.  jusus,  C.  furco,  Peri- 
dinium  divergens,  Prorocentum  micans,  and  Glenodinium  species.  In 
addition  to  brownish-red  and  brownish-yellow  water  extracts,  the 
pigments  of  which  were  regarded  as  analogous  to  the  phycoerythrin 
of  the  Rhodophyceae  and  the  phycophain  of  the  Phaeophyceae,  re- 
spectively, a  wine-red  alcohol  extract  was  obtained.  The  pigment 
thus  extracted,  which  could  not  have  been  pure,  was  soluble  in  ben- 
zene, ether,  chloroform,  carbon  disulfide,  and  glacial  acetic  acid,  but 
very  little  soluble  in  petroleum  ether.  Schiitt  regarded  the  pigment 
as  analogous  to  diatomin,  and  called  it  peridinin.  The  slight  solu- 
bility of  the  pigment  in  petroleum  ether  and  ready  solubility  in  alco- 
hol suggests  that  a  xanthophyll-like  pigment  predominates  in  the 
Peridinieae.    It  is  not  possible  to  draw  more  specific  conclusions  than 


CAROTINOIDS  IN  THE  CRYPTOGAMS  109 

this  on  such  meager  data.  The  writer  is  of  the  opinion  that  exami- 
nation will  disclose  the  fact  that  fucoxanthin  or  a  similar  pigment  is 
the  predominating  carotinoid  in  the  Peridinieae. 

The  Flagellata.  The  flagellates  are  simple  unicellular,  aquatic  or- 
ganisms intermediate  between  the  algae  and  protozoa.  They  inhabit 
ponds  and  streams.  Only  a  few  species  have  been  examined  for  pig- 
ments. A  survey  of  the  somewhat  scanty  evidence  does,  however, 
point  with  certainty  to  the  presence  of  carotinoids  in  those  species 
which  have  been  examined.  The  exact  character  of  the  carotinoids 
remains  to  be  determined. 

Wille  (1887)  regarded  the  pigment  in  the  brown  palmella-like  cells 
of  Chromulina  (Chromophyton)  Rosanoffii  as  diatomin  because  the 
cells  turned  green  when  treated  with  HCl.  Klebs  (1893)  expressed 
the  same  idea  for  Chrysomonidina  Stein,  but  called  the  pigment 
chrysochrome.  Gaidukov  (1900),  however,  emphatically  denied  the 
existence  of  either  carotin  of  fucoxanthin  in  Chromulina,  claiming  to 
have  found  only  two  pigments  present,  a  chlorophyll-like  pigment 
(chrysochlorophyll)  and  a  xanthophyll-like  pigment  (chrysoxantho- 
phyll).  The  latter  pigment  as  described  by  Gaidukov  shows  true 
xanthophyll  properties, 'except  that  only  one  spectroscopic  absorption 
band  was  observed,  namely,  at  495-485[i,[i,  which  corresponds  fairly 
well  with  the  first  xanthophyll  band.  Spectroscopic  studies  of  alco- 
holic and  petroleum  ether  extracts  of  Hydrurus  penicillatus  were 
made  by  Nebelung  (1878),  but  his  results  give  very  little  hint  as  to 
the  true  character  of  the  carotinoids  present. 

Two  species  of  Flagellata  whose  pigment  has  long  been  of  interest 
are  Euglena  sanguinea  and  Euglena  viridis.  In  these  organisms  the 
pigment  occurs  in  a  red  ring  around  the  nucleus,  giving  the  appear- 
ance of  an  eye,  from  which  the  popular  name,  eye-spots,  of  Euglena, 
is  derived.  When  these  organisms  turn  green  the  chlorophyll  develops 
first  at  the  periphery  of  the  red*  ring  and  gradually  spreads  inward. 
The  red  pigment  does  not  always  occur  in  Euglena  sanguinea,  how- 
ever, and  its  absence  seems  to  exert  little  if  any  effect  on  the  normal 
development  of  the  organisms.  The  eye-spots  occur  chiefly  in  spring 
and  autumn,  or  when  the  organisms  are  in  a  dry  state  or  exposed  to 
bright  sunlight.  Cohn  (1850)  and  Klebs  (1883)  regarded  the  red 
coloring  matter  as  identical  with  that  of  the  Chlorophyceae,  Haema- 
tococcus  pluvialis,  the  so-called  haematochrome  of  Cohn.  If  this  be 
the  case  the  eye-spot  pigment  is  a  mixture  of  carotinoids,  inasmuch  as 
Zopf  (1892a)  has  shown  that  haematochrome  consists  of  carotin  and 


no  CAROTINOIDS  AND  RELATED  PIGMENTS 

a  red  xanthophyll-like  pigment  whose  exact  relation  to  the  caro- 
tinoids  remains  to  be  determined. 

The  red  pigment  of  Euglena  sanguinea  was  first  isolated  by  v. 
Wittich  (1863)  and  later  by  Garcin  (1889)  and  Kutscher  (1898). 
V.  Wittich  obtained  microscopic,  garnet  colored  octahedral  crystals  by 
concentrating  the  hot  alcoholic  extract  or  by  adding  alcohol  to  the 
concentrated  ether  solution  of  the  pigment.  The  crystals  were  quickly 
bleached  by  chlorine  and  gave  a  blue  color  reaction  with  concentrated 
sulfuric  acid.  The  crystals  melted  indefinitely  between  70°  and  120° 
C.  They  dissolved  in  hot  alkali  and  the  pigment  could  be  recovered 
from  this  solution  in  amorphous  form,  but  without  loss  of  other  prop- 
erties, by  addition  of  acid.  Neither  Garcin  nor  Kutscher  was  able 
to  extract  the  pigment  from  Euglena  cultures  with  cold  alcohol,  but 
the  former  obtained  orange-red  extracts  with  chloroform,  following 
alcohol  treatment,  and  the  latter  with  boiling  absolute  alcohol.  The 
pigment  extracted  by  Garcin  showed  no  absorption  bands,  but  dis- 
solved in  concentrated  sulfuric  acid  with  a  blue  color.  Garcin  pro- 
posed the  name  rufin  for  the  pigment.  Kutscher's  absolute  alcohol 
extracts  deposited  garnet  colored  crystals  on  concentration.  The 
pigment  as  described  further  by  Kutscher  does  hot  seem  to  be  a  caro- 
tinoid  because  the  recrystallized  substance  melted  at  105°  C.  and 
exhibited  no  absorption  bands.  The  crystalline  pigment,  as  well  as 
its  alcoholic  solution,  turned  blue  on  addition  of  dilute  (50  per  cent) 
sulfuric  or  nitric  acids,  but  alkalies  had  no  ejffect. 

Besides  these  more  critical  studies  Krukenberg  (1886)  found  that 
saponified  alcoholic  extracts  of  Euglena  would  yield  a  greenish-yellow 
lipochrome  to  petroleum-ether  or  ether  in  addition  to  the  red  pigment 
which  acetic  ether  only  would  extract  from  the  soap.  The  red  pig- 
ment, according  to  Krukenberg,  showed  one  absorption  band,  which 
is  contrary  to  the  statement  of  the  other  investigators.  The  greenish- 
yellow  pigment  may  have  been  a  true  carotinoid  inasmuch  as  evi- 
dence of  carotinoids  in  Euglena  was  obtained  by  van  Wisselingh 
(1915)  who  secured  a  positive  Molisch  carotinoid  test  on  the  eye 
spots.  The  chief  pigment  present,  namely,  the  red  one,  does  not, 
however,  appear  to  be  identical  with  any  of  the  known  carotinoids, 
but  resembles  more  nearly  the  red  carotinins  of  Zopf. 

The  Myxophycece  (CyanophyceoB) .  These  constitute  a  large  class 
of  unicellular  or  filamentous  algae  without  a  true  nucleus,  which 
inhabit  both  fresh  and  salt  water  and  are  also  found  in  damp  soil, 
or  on  damp  rocks  and  tree-trunks,  forming  dark  blue-green  patches. 


CAROTINOIDS  IN  THE  CRYPTOGAMS  111 

The  algae  frequently  live  in  symbiosis  with  fungi  or  other  plants. 
Their  characteristic  color  gives  them  their  common  name,  the  blue- 
green  algae.  They  are  among  the  lowest  forms  of  plant  life  which 
are  known. 

The  pigments  of  the  blue-green  algae  have  attracted  the  attention 
of  a  number  of  investigators  beginning  with  Nageli  (1849)  who  ex- 
pressed the  belief  that  these  organisms  contain  a  special  pigment, 
which  he  called  phycochrome,  present  in  two  modifications,  a  blue- 
green  phycocyan,  and  an  orange  phycoxanthin.  It  was  thus  that  the 
latter  name,  later  applied  to  the  pigment  of  the  Phaeophycem,  had 
its  origin.  The  various  species  of  blue-green  algae  which  have  been 
examined  for  carotinoid  pigments  are  given  in  Table  11. 

Table  11.    Myxophyceae  Found  to  Contain  Carotinoids 

Order  Rivulariacece  (highest  forms). 

Calothrix  species — Kraus  and  Millardet,  1868. 

Rivularia  species — Kohl,  1902. 
Order  Scytonemacece. 

Tolypothrix  species — Kohl,  1902. 
Order   Nostocacea. 

Nostoc  species — Kraus  and  Millardet,  1868;  van  Wisselingh,  1915. 

Nodularia — van  Wisselingh,  1915. 

Anabaena  flos  aquce  Bub. — Tammes,  1900;  van  Wisselingh,  1915. 
Order  Oscillatoriacece. 

Oscillatoria—Sorhy,  1873;  Reinke,  1876;  Monteverde,  1893. 

Oscillatoria  {Oscillaria)  limosa — Kraus  and  Millardet,  1868;  Kraus,  1872. 

Oscillatoria  laptotricha — Molisch,  1896. 

Oscillatoria  Froelichii— Tammes,  1900;  Kohl,  1902. 

Phormidium  vulgare — Nebelung,  1878;  Kohl,  1902. 
Order  Chroococcaceoe  (lowest  forms). 

Microcystis  (Polycystis)  flos  aquce  Wittr.  (fresh  water) — Zopf,  1900. 

A  survey  of  the  observations  of  the  various  investigators  shows 
conclusively  that  carotinoids  are  normal  constituents  of  the  Myxo- 
phyceae. This  has  been  demonstrated  microchemically  by  Molisch 
(1896),  Tammes  (1900),  Kohl  (1902)'  and  van  Wisselingh  (1915). 
Spectroscopic  studies  were  made  by  Reinke  (1876)  and  Nebelung 
(1878)  on  different  species,  and  while  the  results  indicate  carotinoids, 
the  solutions  examined  were  not  free  from  other  pigments. 
•  The  evidence  regarding  the  character  of  the  carotinoids  present  is 
less  conclusive.  Kraus  and  Millardet  (1868)  found  that  alcoholic 
extracts  of  Oscillatoria,  Nostoc  arid  Calothrix  species  responded  to  the 
alcohol-benzene  separation,  leaving  a  yellow  pigment  behind  in  the 
alcohol.  Nageli's  designation,  phycoxanthin,  was  adopted  for  the 
pigment  remaining  in  the  alcohol.  Kraus  (1872),  however,  later  rec- 
ognized that  more  than  one  pigment  was  probably  present  in  these 


112  CAROTINOIDS  AND  RELATED  PIGMENTS 

algae,  and  expressed  the  belief  that  the  pure  xanthophyll-like  phyco- 
xanthin  in  Oscillatoria  is  closely  related  to,  but  not  identical  with,  the 
xanthophyll  of  higher  green  plants. 

Sorby  (1873)  differentiated  between  phycoxanthin,  fucoxanthin  and 
"orange  xanthophyll"  (carotin)  in  Oscillatoria.  Sorby's  phycoxanthin 
is  not  identical  with  the  so-called  phycoxanthin  of  brown  algae,  the 
pigment  now  known  as  fucoxanthin.  As  described  by  Sorby  phyco- 
xanthin is  practically  non-extractable  from  alcohol  by  carbon  disul- 
fide, but  when  dissolved  in  the  latter  solvent  gives  red  solutions, 
which  are  still  pink  in  great  dilution,  the  absorption  bands  in  this 
solvent  being  shifted  towards  the  red  end  of  the  spectrum  to  even  a 
greater  extent  than  those  of  carotin.  The  presence  of  such  a  pigment 
in  Oscillatoria  has  not  been  reported  by  others,  and  its  relation  to  the 
carotinoids  remains  to  be  determined.  Sorby's  fucoxanthin  is  identi- 
cal with  the  fucoxanthin  of  the  brown  algae,  whose  chemical  prop- 
erties have  been  described.  No  other  investigator  has  reported  the 
presence  of  this  pigment  in  the  blue-green  algae.  If  Sorby's  observa- 
tions can  be  substantiated  it  will  show  that  this  pigment  is  much 
more  universally  distributed  among  the  algae  than  has  been  hereto- 
fore regarded. 

Sorby  also  reported  observations  regarding  the  distribution  of 
phycoxanthin,  ''orange  xanthophyll"  and  fucoxanthin  in  Oscillatoria 
with  different  exposures  of  the  organisms  to  light,  finding  that  the 
more  intense  the  light  during  growth  the  more  phycoxanthin  and 
"orange  xanthophyll"  (carotin)  they  contain  and  the  less  fucoxanthin. 

Further  evidence  regarding  the  presence  of  carotin  in  the  blue- 
green  algae  was  furnished  by  Monteverde  (1893),  who  demonstrated 
carotin  in  Oscillatoria  by  the  Kraus  method.  The  question  of  the 
character  of  the  pigments  remaining  in  the  alcohol  following  the 
separation  between  this  solvent  and  petroleum  ether  was  left  open 
by  Monteverde. 

That  even  the  lowest  forms  are  abundantly  pigmented  by  carotin 
has  been  shown  by  Zopf  (1900)  who  has  described  the  ease  with  which 
carotin  crystals  can  be  obtained  from  Microcystis  (Polycystis)  flo's 
aqucB  Wittr.  It  is  doubtful,  however,  whether  Zopf  is  justified  in  re- 
garding the  carotin  as  a  special  pigment,  and  ascribing  to  it  the  name 
polycystin. 


CAROTINOIDS  IN  THE  CRYPTOGAMS  113 

Carotinoids  in  the  Fungi 

The  brilliancy  of  color  which  characterizes  practically  all  classes 
of  fungi  is  a  fact  which  is  familiar  even  to  the  layman  in  the  fields 
of  botany  and  biology.  Yellow,  orange  and  red  colors  are  by  no 
means  the  least  conspicuous  among  these  plants,  and  may  in  many 
cases  be  regarded  as  the  predominating  ones.  This  fact,  together 
with  the  absence  of  chlorophyll  from  this  form  of  plant  life,  is  what 
gives  prominence  to  a  consideration  of  the  relation  of  the  pigments 
involved  to  pigments  of  similar  color,  namely,  carotinoids,  produced 
in  the  chlorophyllous  plants.  As  has  been  already  pointed  out,  how- 
ever, yellow,  orange  and  red  colors  in  fungi  appear  to  be  more  fre- 
quently non-carotinoid  in  nature  than  possessing  the  characteristics 
of  the  chromolipoids.  Zopf  (1890)  mentions  a  number  of  instances 
where  this  is  the  case.  Nevertheless,  carotinoids  do  occur  among  the 
fungi,  especially  among  the  higher  forms,  and  the  evidence  for  this 
conclusion  will  now  be  presented.  It  will  be  apparent,  however,  that 
specific  evidence  is  almost  completely  lacking  as  to.  the  kinds  of  caro- 
tinoids involved.  The  plan  of  presentation  will  be  similar  to  that 
followed  in  the  case  of  the  algae. 

The  Basidiomycetes.  The  various  fungi  which  comprise  this  group 
include  the  numerous  species  of  mushrooms,  toadstools  and  bracket 
fungi  (included  together  under  the  Hymenomycetes) ,  the  puff-balls 
(Gasteromycetes)  known  to  every  school  child,  the  rusts  (Uredineae) , 
and  the  smuts  (Ustilaginece) .  Yellow  colors  do  not  especially  char- 
acterize the  Gasteromycetes,  and  so  far  as  the  author  is  aware  caro- 
tinoids have  not  been  demonstrated  in  any  of  the  members  of  this 
family.  The  same  statement  likewise  holds  true  for  the  smuts. 
Yellow  to  orange-red  tints  are  very  common,  however,  among  the 
Hymenomycetes,  and  the  Uridineae  take  their  common  name  (rusts) 
from  the  predominating  color  of  their  spores. 

The  species  of  Basidiomycetes  which  may  be  regarded  as  owing 
their  color  to  carotinoids  or  related  pigments  are  collected  in  Table 
12.  It  is  not  to  be  considered  that  this  short  list  comprises  all  the 
species  which  probably  contain  carotinoids,  but  merely  that  proof  has 
been  furnished  for  those  mentioned.  For  example,  the  common  mush- 
room Clavaria  fusiformis  (Golden  Spindle)  may  owe  its  color  in  part 
to  carotinoids  although  Sorby  (1873)  in  his  study  of  the  pigments  of 
many  classes  of  plants  speaks  of  this  fungus  only  as  a  source  of 


114  CAROTIN OID&  AND  RELATED  PIGMENTS 

"lichnoxanthin,"  whose  exact  relation  to  known  pigments  is  as  yet 
obscure. 

All  of  the  Hymenomycetes  which  are  known  to  contain  carotinoids 
are  bracket  fungi  which  grow  on  decaying  wood  or  among  fallen 
leaves.  Whether  these  fungi  derive  their  carotinoids  from  the  hosts 
upon  which  they  grow  or  synthesize  their  own  pigments  remains  to 
be  determined. 

Calocera  viscosa  is  a  very  sticky  fungus  of  a  beautiful  orange  color 
which  is  found  abundantly  on  rotten  tree  stumps,  especially  fir,  in 
the  autumn.  It  grows  one  to  three  inches  high.  C.  cornea  is  not  so 
highly  colored  and  grows  in  spikes  one-fourth  to  two-thirds  inches 
high  on  dead  wood.  Dacromyces  stillatus  forms  deep  orange  colored 
spots  on  pine  and  other  decaying  wood.  Ditiola  radicata  produces  a 
golden-yellow  hymenium  two  to  three  inches  across  on  rotten  wood, 
and  among  fallen  pine  leaves,  etc. 

Table  12.    Basidiomycetes  Found  to  Contain  Carotinoids 

Hymenomycetes. 

Calocera  viscosa — Zopf,  1889c;  van  Wisselingh,  1915. 

Calocera  cornea — ^van  Wisselingh,  1915. 

Calocera  palniata — van  Wisselingh,  1915. 

Dacryomyces  stillatus — Zopf,  1889c. 

Ditiola  radicata — Zopf,  1893a. 
UredinecB  (rusts). 

Gymnosporangium  juniperinum — Bachmann,  1886. 

Melampsora  Salicis  caprece — Bachmann,  1886. 

Puccinia  coronata — Bachmann,  1886. 

Triphragmium  Ulmarice — Bachmann,  1886. 

Uromyces  alchemillce — Bachmann,  1886. 

Coleosporium  Pulsatilla  Strauss — Muller,  1885;  Bertrand  and  Poirault,  1892. 

Uredo  euphrasix — Bertrand  and  Poirault,  1892. 

Melampsora  aecidioides  D.  C. — Muller,  1886;  Bertrand  and  Poirault,  1892. 

Phragmidium  violaceum — Muller,  1886. 

Aecidia,  Promycelia  and  Sporidia  Spores — Kohl,  1902. 

The  evidence  that  these  fungi  owe  their  color  to  carotinoids  was 
first  furnished  by  Zopf  (1889c)  who  found  that  the  chromolipoid 
when  isolated  not  only  responded  to  the  blue  color  reaction  with  con- 
centrated sulfuric  acid,  which  Zopf  called  the  lipocyan  reaction,  but 
could  be  made  to  produce  blue  microscopic  crystals  under  the  influ- 
ence of  this  reagent.  Zopf  described  in  detail  the  method  for  the 
formation  of  the  blue  "lipocyan"  crystals  for  the  pigments  of  these 
fungi  and  also  for  other  lipochrome  containing  materials.  More 
conclusive  evidence  that  these  fungi  owe  their  color  to  carotinoids 
was  furnished  by  Zopf  (1893a)  who  isolated  a  pigment  showing  the 
spectroscopic  absorption  bands  of  carotin  from  Ditiola  radicata,  and 


CAROTINOIDS  IN  THE  CRYPTOGAMS  115 

more  recently  by  van  Wisselingh  (1915)  who  was  able  to  secure 
crystals  of  pigment  by  the  Molisch  microchemical  test.  The  latter 
investigator  states  that  about  twenty-five  fungi  which  he  examined 
by  this  method  failed  to  respond  to  the  test,  but  mentions  specifically 
only  those  which  responded.  Special  attention  was  given  to  the  crys- 
tals produced  in  the  case  of  Calocera  viscosa  and  Dacryomyces 
stillatus.  From  the  former  a  heavy  precipitation  of  orange  colored 
crystals  was  secured  from  a  section  between  the  hypens.  These  gave 
all  the  carotinoid  color  reactions,  and  the  crystals  dissolved  slowly  in 
phenol-glycerin,  indicating  a  xanthophyll-like  pigment.  C.  cornea 
and  C.  palmata  gave  like  results.  In  the  case  of  Dacryomyces  stil- 
latus crystals  of  a  similar  color  were  secured,  as  well  as  red  colored 
crystals  and  orange-yellow  aggregates,  suggesting  the  possibility  of 
several  carotinoids  being  present. 

Particularly  interesting  is  the  abundant  evidence  that  the  coloring 
matter  of  the  rust  fungi  is  carotinoid  in  nature.  Bachmann  (1886) 
first  called  attention  to  the  fact  that  the  rusts  from  several  different 
hosts  owe  their  color  to  orange  or  yellow  oil  globules  containing 
unsaponifiable  chromolipoids.  The  pigment  was  isolated  by  cutting 
out  the  rust  spots,  extracting  them  with  ether  or  hot  alcohol,  saponify- 
ing the  extract,  and  extracting  the  soap  with  petroleum  ether.  The 
positions  of  the  spectroscopic  absorption  bands  of  the  extracts  thus 
obtained  correspond  closely  with  those  of  carotin.  The  residues  from 
the  extracts  gave  the  usual  color  reactions  with  concentrated  sulfuric 
acid  and  iodine.  Miiller  (1886)  observed  that  red  pigment  crystals 
appeared  in  the  spores  of  certain  rusts  when  placed  in  glycerin. 
Zopf  (1890)  held  that  these  were  due  to  a  pigment  other  than  lipo- 
chrome,  but  that  the  fungi  contain  the  lipochrome  as  well  as  the 
special  red  pigment.  Bertrand  and  Poirault  (1892),  however,  who 
observed  the  same  phenomenon  in  the  rusts  examined  by  Miiller,  as 
well  as  other  species,  regarded  the  red  crystals  to  be  due  to  cholesterol 
colored  by  carotin,  inasmuch  as  identical  crystals  are  formed  when  the 
pollen  grains  of  Verbascum  thapsiforme  L.  (mullein)  are  mounted 
in  glycerin.  In  view  of  the  observations  of  Bachmann  pointing  con- 
clusively to  the  presence  of  carotinoids  in  the  rusts,  it  seems  likely 
that  pigments  of  this  type  are  involved  in  the  crystals  observed  by 
Miiller  and  by  Bertrand  and  Poirault.  It  should  be  noted,  however, 
that  the  uredo  spores  of  these  fungi,  which  is  the  chief  pigmented 
stage  in  their  development,  are  not  colored  alike  for  the  various 
species,  the  color  varying  from  yellow  to  reddish  orange.    The  winter 


116  CAROTINOIDS  AND  RELATED  PIGMENTS 

stage,  or  teleutospores,  is  usually  black,  but  in  the  case  of  one  of  the 
species  mentioned  in  Table  12,  namely,  Uredo  euphrasix  Schum.,  this 
stage  is  red.  Other  stages  in  the  life  cycle  of  the  rusts  appear  to  con- 
tain carotinoids  also,  since  Kohl  (1902)  reports  a  positive  Molisch 
test  on  promycelia,  sporidia  and  secidia  of  the  fungi,  the  consecutive 
stages  in  the  germination  of  the  teleutospores  to  the  uredospore  stage. 
The  Ascomycetes.  These  fungi,  which  are  commonly  known  as  the 
cup  or  sac  fungi  because  of  their  shape,  are  frequently  brilliantly 
colored  with  yellow,  orange  or  reddish  pigments.  The  coloring  mat- 
ter responsible  for  these  tints  has  naturally  attracted  the  attention 
of  a  few  of  the  pigment  workers,  notably  Zopf,  to  whom  we  owe  much 
of  our  knowledge  regarding  the  fungi  pigments.  The  species  of 
Ascomycetes  whose  pigmentation  may  with  some  assurance  be  re- 
garded as  due  largely  to  carotinoids  are  mentioned  in  Table  13.  No 
doubt  others  could  be  added  to  the  list. 

Table  13.    Ascomycetes  Found  to  Contain  Carotinoids 

Discomycetes. 

Peziza  aurantia — Zopf,  1892b. 

Peziza  bicolor  Biill. — Bachmann,  1886. 

Peziza  scuteUata  L. — Bachmann,  1886. 

Leotia  lubrica—Zopi,  1890,  1892b;  Kohl,  1902. 

Ascobolus  species — Zopf,  1889c,  1892b. 

Spathularia  flauida  Pers.— Zopf,  1892b;  Kohl,  1902. 
Pyrenomycetes. 

Polystigma  rubrum — Zopf,  1893a. 

Polystigma  ochraceum  Wahlenberg  (=  P.  julvuni  D.  C.) — Zopf,  1893a. 

Spaerostilbe  coccaphila — van  Wissehngh,  1915. 

Nectria  cinnabarina — Bachmann,  1886;   Zopf,  1893a;   Kohl,  1902;   van  Wis- 
sehngh, 1915. 

The  Peziza  genera  of  the  Discomycetes  contains  several  species  with 
especially  bright  color.  Peziza  aurantia,  which  is  sometimes  called 
"orange-peel  Elf-cup"  takes  the  form  of  a  shallow,  irregular  shaped 
cup,  one  to  three  inches  in  diameter,  and  resembles  closely  a  piece  of 
inverted  orange  peel.  The  outside  of  the  fungus  is  pale  orange  but 
the  interior  is  a  brilliant  orange  or  orange  red.  It  is  frequently  found 
on  the  flat  ground  in  autumn.  Peziza  bicolor  Blill.  forms  a  yellow  to 
deep  orange-red  disc  on  dead  branches  of  oak,  hazel  and  hawthorn 
trees,  and  P.  scuteUata  L.  a  deep  carmine  colored  disc  on  rotten  tree 
stumps.  Sorby  (1873)  examined  the  pigment  of  the  first  mentioned 
species  but  called  the  pigment  peziza  xanthin  and  did  not  identify  it 
with  the  "xanthophylls"  of  the  higher  plants  or  the  algae.  Bachmann 
(1886)  first  showed  the  presence  of  chromolipoids  in  the  Peziza  fungi 
when  he  recovered  unsaponifiable  pigment  from  them  showing  the 


CAROTINOIDS  IN  THE  CRYPTOGAMS  117 

spectroscopic  absorption  bands  and  color  reactions  of  the  lipochromes 
from  higher  plants.  This  was  confirmed  by  Zopf  (1892b)  for  Peziza 
aurantia,  who  at  the  same  time  reported  lipochromes  in  two  other 
Discomycetes,  namely,  -Leotia  lubrica  and  Spathularia  flavida  Pers., 
but  was  unable  to  find  lipochrome  in  Bulgaria  inquinans  {=poly- 
morpha) .  The  presence  of  carotinoids  in  the  two  first  mentioned  was 
later  confirmed  by  Kohl  (1902)  using  the  Molisch  test.  Leotia 
lubrica,  however,  owes  its  color  in  part  to  a  green  colored  pigment  as 
well  as  to  chromolipoid  (Zopf,  1890).  It  is  of  interest  to  note  also 
that  Zopf  (1889c,  1892b)  reported  that  carotinoid-like  pigments  could 
be  isolated  from  various  species  of  Ascobolus,  which  flourish  on  the 
feces  of  animals. 

Among  the  Pyrenomycetes  Bachmann  (1886)  first  reported  unsap- 
onifiable  lipochrome  in  Nectria  cinnabarina  (Tode)  Fries.,  which  was 
later  studied  in  detail  together  with  the  pigments  of  Polystigma  rub- 
rum  Pers.  and  P.  ochraceum  (=  P.  fulvum  D.  C.)  by  Zopf  (1890, 
1893a).  The  former  is  a  cushion  shaped,  red  fungus  found  on  the 
dead  branches  of  deciduous  trees,  while  Polystigma  attack  the  foliage 
of  plum  trees,  forming  red  or  red-brown  spots  on  the  leaves. 

The  lipochrome  which  Bachmann  isolated  from  Nectria  cinnabarina 
corresponded  in  spectroscopic  bands  with  xanthophyll.  A  red  resin 
was  also  reported  in  this  fungus.  Zopf  used  the  conidial  layer  of  the 
fungus  obtained  from  Aesculus  Hippocastanum  for  his  study.  The 
presence  of  a  two-banded  "carotin"  was  confirmed  and  the  red  resin 
of  Bachmann  was  found  to  conform  to  a  number  of  other  "carotinins" 
studied  by  this  investigator  (e.g.,  the  red  pigment  of  Haematococcus 
pluvialis  already  discussed)  in  that  it  readily  formed  compounds  with 
sodium  and  barium.  The  sodium  salt  was  practically  insoluble  in 
alcohol  and  ether,  but  soluble  in  chloroform,  benzene  and  carbon 
disulfide,  and  the  barium  salt  was  insoluble  in  all  these  solvents.  The 
ethereal  solution  of  the  base-free  pigment  showed  two  bands  at  512- 
490[in  and  481-464}X[i,  the  solution  in  carbon  disulfide  showing  three 
bands  at  575-553[X[x,  530-508[X[i  and  494-482(X[i.  The  relation  of  this 
pigment  to  the  carotinoids  remains  to  be  determined.  It  was  either 
this  pigment  or  the  yellow  "carotin"  which  responded  to  the  Molisch 
test  in  the  hands  of  Kohl  (1902)  and  van  Wisselingh  (1915).  Zopf 
called  the  red  pigment  nectriin  or  nectria  red. 

Zopf  found  two  pigments  in  Polystigma  rubrum  which  were  .very 
similar  to  those  in  N.  cinnabarina,  the  absorption  spectra  of  the 
"carotin"  indicating  identity  with  the  carotin  of  carrots,  the  red  pig- 


118  CAROTINOIDS  AND  RELATED  PIGMENTS 

ment,  which  appears  to  be  the  chief  one  present,  differing  from  nec- 
triin  in  the  position  and  number  of  the  absorption  bands  (polystig- 
min,  as  Zopf  calls  it,  showing  only  two  even  in  carbon  disulfide),  and 
also  in  that  the  barium  compound  is  soluble  in  ether,  chloroform,  car- 
bon disulfide  and  alcohol.  Zopf's  examination  of  Polystigma  ochra- 
ceum,  which  has  more  of  a  yellow  than  a  red  color,  showed  an  abun- 
dance of  yellow  "carotin,"  which  was  regarded  as  produced  in  the 
fungus  cells.  No  red  pigment  was  found,  but  the  fungus  was  not  de- 
colorized after  the  extraction  of  the  carotinoid,  but  was  left  a  reddish- 
brown  color  which  could  be  extracted  by  dilute  ammonium  hydroxide. 

Van  Wisselingh  (1915)  made  a  special  examination  of  the  micro- 
chemical  crystals  formed  in  Spaerostilbe  coccaphila,  a  red  fungus 
found  on  fallen  trees.  The  fungus  itself  contains  red,  fat-like  globules. 
Violet-red  crystals  were  produced  in  the  Molisch  test,  which  gave  the 
carotinoid  color  reactions  and  dissolved  readily  in  the  phenol-glycerin 
reagent  which  appears  to  be  specific  for  xanthophyll. 

The  Phycomycetes.  This  class  of  fungi  includes  the  molds,  the 
mildews  and  the  yeasts  and  thus  contains  many  species  of  plants  of 
great  importance.  One  does  not  ordinarily  associate  carotinoid  colors 
with  these  fungi,  and  the  presence  of  such  pigments  does  not,  in  fact, 
appear  to  be  common.  Carotinoids  have  been  demonstrated  to  be 
present,  however,  in  several  instances. 

Zopf  (1892b)  was  able  to  extract  a  carotinoid  from  three  species 
of  Pilobolus,  namely,  P.  crystallinus,  P.  Kleinii  and  P.  Oedipus,  which 
gave  the  lipocyan  reaction,  the  lipochrome  reaction  with  iodine,  and 
also  showed  absorption  bands  in  petroleum  ether  at  484-469[X|j,  and 
452-439jx[x,  which  correspond  closely  with  xanthophyll.  The  first  two 
species  flourish  on  fresh  horse  dung,  the  last  on  dung  or  rotting  algae. 
Zopf  also  stated  (1892b)  that  Pleotrachelus  fulgens,  a  reddish-brown 
species  of  another  order,  is"  a  carotin  (oid)  former,  but  the  evidence 
for  this  was  not  presented. 

Kohl  (1902)  confirmed  the  presence  of  carotinoids  in  Philobolus 
species  using  the  Molisch  test,  and  also  showed  the  same  pigments  to 
be  present  in  Mucor  species  and  in  Chytridium. 

Van  Wisselingh  (1915)  included  Mucor  flavus  Bainer  in  his  micro- 
chemical  studies.  Orange-yellow  crystals  were  secured  by  the  Molisch 
method,  which  gave  the  carotinoid  color  reactions  with  nitric  acid 
and  with  bromine. 

The  Myxomycetes.  These  fungi  form  a  distinct,  independent  group 
of  plants,  commonly  known  as  slime  molds,  and  were  formerly  classi- 


CAROTIN OWS  IN  THE  CRYPTOGAMS  119 

fied  in  the  animal  kingdom  under  the  name  Mycetozoa.  The  plants 
are  naked  masses  of  protoplasm,  called  plasmodia,  which  exhibit  many 
beautiful  colors  as  shown  in  Lister's  well-known  work  on  the  slime 
molds.  Carotinoids  are  probably  rare  in  this  group,  although  this 
statement  may  be  hasty  inasmuch  as  very  few  species  have  been 
examined.  Carotinoids  do  not  appear  to  be  present  in  Arcyria  punicea 
Pers.  and  Ar.  nutans  Biill.  or  in  Aethalium  septicum  Fr.,  a  species  of 
Fuligo  Septica — the  well-known  "Flowers  of  Tan" — according  to  the 
observations  of  Schroeter  (1875),  Zopf  (1892b)  and  Bachmann  (1886). 
Carotinoids  do  appear  to  be  present,  however,  in  Stemonitis  ferru- 
ginea,  Stemonitis  jusca,  Lycogala  epidendron  and  Lycogala  flavo- 
juscum,  judging  from  the  observations  of  Zopf  (1889b)  who  made  a 
special  study  of  the  possible  presence  of  lipochromes  in  Myxomycetes. 
In  no  case  was  lipochrome  found  to  be  the  only  pigment  present, 
although  absolute  alcohol  was  found  to  extract  completely  the  color 
from  the  carrot-red  plasmodia  and  fruits  of  L.  epidendron.  In  each 
case  an  unsaponifiable  lipochrome  was  isolated  showing  the  color 
reaction  with  concentrated  sulfuric  acid.  The  measurements  of  the 
position  of  the  absorption  bands  of  the  lipochrome  of  each  species  as 
reported  by  Zopf  indicate  a  xanthophyll-like  pigment  in  the  case  of 
Stemonitis,  but  carotin  in  the  case  of  Lycogala.  These  observations 
might  well  be  amplified  by  others,  carried  out  in  the  light  of  our 
present  knowledge  of  the  carotinoids. 

The  Imperfect  Fungi.  There  is  evidence  that  carotinoids  are  pres- 
ent in  a  few  species  of  this  large  group  of  fungi  whose  exact  classi- 
fication has  not  yet  been  determined. 

Zopf  (1889c)  states  that  the  pigment  which  can  be  extracted  with 
fat  solvents  from  Cephalothecium  gives  the  blue  lipocyan  crystals 
with  sulfuric  acid.  Several  of  the  fungi  which  gave  positive  evidence 
of  carotinoids  microchemically  in  van  Wisselingh's  study  (1915)  be- 
long in  the  group  of  imperfects.  For  example,  Monilia  sitophila 
(Mont.)  Dace,  gave  red  crystals  in  the  Molisch  test;  Aspergillus 
giganteus,  which  has  an  orange-yellow  mycelium,  gave  a  positive  test; 
Torula  rubra  also  gave  the  reaction,  but  Torula  cinnabarina  failed  to 
do  so,  although  a  color  reaction  was  secured  using  SbClg. 

Carotinoids  in  Bacteria 

The  importance  of  bacteria  as  a  means  of  determining  some  of  the 
true  functions  of  carotinoids  in  plants,  or  at  least  of  fixing  the  con- 


120  CAROTINOIDS  AND  RELATED  PIGMENTS 

ditions  under  which  they  develop,  has  already  been  pointed  out.  The 
work  upon  which  our  present  knowledge  of  carotinoids  in  bacteria  is 
based  will  now  be  reviewed.  Bacteria  are  at  present  classified  as 
Schizomycetes,  and  are  best  considered  as  algae.  Their  morphology 
and  reproduction  most  nearly  resemble  the  Cyanophyceae.  In  fact, 
bacteria  are  considered  by  some  as  having  "degenerated"  from  the 
blue-green  algae.  They  do  not,  however,  contain  chlorophyll,  and  it 
is  this  fact,  especially,  which  enhances  the  interest  in  the  possibility 
of  carotinoids  being  normal  constituents  of  these  organisms. 

As  in  the  case  of  non-chlorophyll  bearing  fungi,  it  is  not  to  be 
assumed  that  all  yellow,  orange  and  red  tinted  bacterial  colonies  owe 
their  color  to  carotinoids.  The  pigments  of  B.  prodigiosus  and  B. 
xanthinum  Ehren.  first  described  by  Schroeter  (1875)  are  obviously 
not  carotinoids,  although  color  alone  would  suggest  that  this  is  the 
case.  Grifiiths  (1892)  ascribes  the  formula  CssHsgNOg  to  the  red 
pigment  of  B.  prodigiosus,  but  the  empirical  relation  between  the  car- 
bon and  hydrogen  suggests,  rather  than  negatives  a  relation  of  the 
pigment  to  carotin.  Schroeter  described  the  change  of  color  of  the 
colonies  of  this  bacteria  from  red  to  orange  to  yellow  and  ascribed  it 
to  the  formation  of  an  alkaline  substance  in  the  course  of  the  growth 
of  the  bacteria.  This  variation  in  color  of  B.  prodigiosus  is  probably 
well  known  to  bacteriologists  and  might  be  thought  to  be  due  either 
to  a  variation  in  concentration  of  the  same  pigment  or  to  the  presence 
of  distinct  yellow  (possibly  carotinoid)  and  red  pigments,  the  latter, 
when  present,  masking  the  former.  Schroeter  supported  his  explana- 
tion of  the  change  in  color,  however,  by  showing  that  the  orange-red 
alcoholic  extract  of  the  bacteria  turns  red  with  acid  and  yellow  with 
alkali. 

Aside  from  the  brief  observation  of  Schrotter  (1895)  that  the  pig- 
ments of  Sarcina  aurantiaca  and  M.  (Staph.)  pyrogenes  aureus  show 
the  solubility  properties  and  color  reaction  (with  H2SO4)  of  "lipo- 
xanthin"  (carotinoid)  our  knowledge  regarding  carotinoid  producing 
species  of  bacteria  is  due  apparently  solely  to  Zopf  (1889,  a,  c;  1891; 
1892b)  who  has  described  the  chromolipoids  in  eight  species  of  bac- 
teria. The  descriptions  as  given  by  Zopf  point  with  certainty  to  caro- 
tinoids in  the  case  of  four  species  only,  namely,  B.  egregium,  B.  Chry- 
sogloia,  M.  (Staph.)  aureus  and  Sphaerotilus  roseus.  The  first  three 
of  these  bacteria  form  yellow  colonies,  but  the  last  mentioned  species 
is  red.    The  evidence  for  carotinoids  is  as  follows: 

B.  egregium.    Form's  intensely  yellow  colonies  on  gelatin  or  beef- 


CAROTINOIDS  IN  THE  CRYPTOGAMS  121 

extract  agar  (b.e.  2.3  per  cent,  agar  one  per  cent).  Colonies  when 
transferred  to  porcelain  plate  give  blue  color  with  concentrated  H2SO4 
and  HNO3,  and  blue  microscopic  crystals  with  former  (lipocyan  reac- 
tion, Zopf).  Pigment  is  slowly  extracted  by  warm  absolute  alcohol 
and  when  thus  extracted  is  soluble  in  alcohol,  ether,  chloroform, 
methyl  alcohol,  benzene  and  petroleum  ether.  The  alcoholic  solutions 
show  two  absorption  bands,  one  covering  the  F  line,  the  other  between 
F  and  G.  The  pigment  is  not  saponifiable.  It  develops  in  the  dark 
as  well  as  in  the  light. 

B.  Chrysogloia.  The  yellow  pigment  produced  corresponds  exactly 
in  properties  with  that  of  B.  egregium. 

M.  (Staph.)  aureus.  The  yellow  pigment  shows  the  same  prop- 
erties described  for  the  above  mentioned  bacteria,  according  to  Zopf. 

Spaerotilu^  roseus.  A  red  bacteria  giving  a  yellow  to  yellowish-red 
alcoholic  extract.  Strips  of  filter  paper  immersed  at  one  end  in  the 
extract  showed  in  time  three  zones,  a  wide  red  zone  over  which  was  a 
narrow  yellow  zone  and  over  this  a  very  narrow  brownish  zone.  The 
yellow  pigment  was  soluble  in  water  and  the  red  one  in  alcohol,  ether, 
chloroform,  ligroin,  petroleum  ether,  benzene  and  carbon  disulfide. 
After  saponification  and  extraction  with  petroleum  ether  the  pigment 
showed  all  the  properties  of  "eucarotin,"  the  absorption  bands  in 
alcohol  lying  at  492-474[i[i  and  456-442|.i[j,.  The  properties  described 
are  strongly  indicative  of  carotin. 

There  is  much  less  certainty  regarding  the  character  of  the  pig- 
ments in  the  other  species  of  bacteria  examined  by  Zopf,  although 
the  pigment  is  ascribed  by  Zopf  to  "lipochrome."  The  red  color  of 
M.  [Staph.)  apatelus  and  M.  (Staph.)  superbus  is  stated  (1889c)  to 
be  a  red  lipochrome  which  gives  the  microscopic  blue  lipocyan  crys- 
tals with  concentrated  H2SO4.  The  red  pigment  of  M.  (Staph.)  rho- 
dochrous  and  M.  (Staph.)  Erythromyxa  gives  the  same  reaction.  Old 
colonies  of  these  two  bacteria  show  scarlet  or  blood-red  crystal  aggre- 
gates under  the  microscope  (dark  field),  according  to  Zopf  (1891). 
These  crystals  are  soluble  in  alcohol,  ether,  chloroform,  petroleum 
ether,  benzene  and  carbon  disulfide  and  these  solutions  are  charac- 
terized by  showing  only  one  wide  absorption  band  in  the  spectroscope 
at  F.  The  pigment  is  not  saponifiable.  It  is  not  clear  whether  this 
pigment  is  one  of  the  known  carotinoids  or  is  to  be  classified  with  the 
red  "carotinins,"  which  have  been  repeatedly  mentioned,  and  the 
determination  of  whose  relation  to  the  carotinoids  is  greatly  to  be 
desired.     Overbeck   (1891),  who  has  studied  the  physiology  of  pig- 


122  CAROTINOIDS  AND  RELATED  PIGMENTS 

merit  production  in  the  two  last  mentioned  species  of  bacteria,  states 
that  M.  Erythromyxa  produces  a  yellow  water-soluble  pigment  in 
addition  to  the  red  lipochrome. 


Summary  , 

Our  knowledge  is  practically  complete  regarding  the  character  and 
distribution  of  the  carotinoids  among  certain  classes  of  algae,  par- 
ticularly the  brown  and  red  sea-weeds. 

Fresh  brown  sea-weeds  owe  their  olive-brown  tint  to  the  special 
algae  carotinoid,  fucoxanthin,  discovered  by  Rosanoff  (1867)  and 
Millardet  (1869),  and  finally  classified  definitely  as  a  carotinoid  by 
Willstatter  and  Page  (1914).  The  relation  of  this  pigment  to  carotin 
is  shown  by  the  empirical  formula  C^oHggOe.  The  characteristic  prop- 
erties of  the  pigment  are  described  in  detail  in  the  text. 

Brown  sea-weeds  also  contain  carotin  and  xanthophyll.  The  exact 
relation  of  this  xanthophyll  to  the  xanthophylls  of  higher  plants  has 
not  been  definitely  settled. 

Dried  brown  sea-weeds  owe  their  color  to  phycophain,  a  post- 
mortal oxidation  product  of  colorless  chromogens  present  in  the 
fresh  plants.  The  carotinoids  are  still  present,  but  the  phycophain 
interferes  greatly  with  their  isolation  and  study. 

The  principal  pigment  of  red  sea-weeds  is  phycoerythrin,  which  is 
not  a  carotinoid.  Carotin  and  xanthophyll,  however,  are  present  in 
these  plants.  There  are  some  indications  that  the  xanthophyll  is  the 
xanthophyll  p  which  characterizes  higher  plants.  There  is  a  possi- 
bility, also,  that  fucoxanthin  is  present  in  the  red  algae, 

Carotinoids  are  present  in  the  stone  worts,  but  nothing  is  known 
of  their  nature. 

Carotin  and  xanthophyll  are  present  in  the  green  algse,  the  amount 
of  each  present  in  certain  species  having  been  determined  by  Will- 
statter and  Page.  The  red  pigment  of  the  so-called  blood  algae  clas- 
sified among  this  family,  appears  to  be  related  to  the  carotinoids,  but 
its  exact  relation  remains  to  be  determined. 

Carotin  appears  to  be  the  principal  carotinoid  present  in  the  di- 
atoms. There  is  a  possibility,  also,  that  xanthophylls  and  fucoxan- 
thin are  present,  a  phase  of  the  pigmentation  of  the  siliceous  algse 
which  deserves  further  study. 

Our  knowledge  is  indefinite  regarding  the  carotinoids  occurring  in 
the  Peridiniales,  although  the  indications  are  that  a  xanthophyll-like 


CAROTINOIDS  IN  THE  CRYPTOGAMS  123 

pigment,  possibly  fucoxanthin,  predominates  among  the  chromolipoids 
present.  Brownish-red  and  yellow  water-soluble  non-carotinoids  are 
the  chief  cause  of  the  color  of  these  plants. 

Carotinoids  are  unquestionably  present  in  the  flagellates,  although 
their  exact  nature  remains  to  be  determined.  The  red  pigment  which 
characterizes  the  so-called  eye-spots  of  Euglena  species  does  not  ap- 
pear to  be  identical  with  any  of  the  known  carotinoids,  but  resembles 
the  red  carotinins,  the  determination  of  whose  relation  to  the  carotin- 
oids is  greatly  to  be  desired. 

Carotinoids  are  normal  constituents  of  the  blue-green  algae.  The 
facts  which  are  known  point  to  the  presence  of  carotin  and  fucoxanthin 
in  these  plants.  Another  pigment  is  present  in  certain  species,  which 
is  non-carotinoid  in  nature  but  which  resembles  carotin  in  having  a 
yellow  color  in  alcohol  and  a  red  color  in  carbon  disulfide.  Its  exact 
nature  is  not  known. 

Carotinoid  colors  are  more  common  among  the  fungi  than  among 
the  algae  but  the  color  in  many  cases  appears  to  be  due  to  other 
pigments.  In  fact,  many  fungi  seem  to  be  entirely  devoid  of  carotin- 
oid pigments. 

Among  the  Basidiomycetes,  a  few  species  in  the  mushroom  family 
apparently  owe  their  color  to  carotinoids.  The  striking  examples  of 
carotinoid  pigmentation,  however,  are  the  rusts,  whose  yellow  and 
red  colors  are  due  to  carotin  or  a  very  closely  related  pigment.  It 
is  not  known  whether  other  carotinoids  are  involved. 

Several  of  the  brilliantly  colored  cup  fungi  owe  their  color  to 
carotinoids.  The  exact  nature  of  these  has  not  been  determined  in 
the  case  of  the  Discomycetes,  but  in  the  case  of  certain  Pyrenomycetes 
carotin  is  undoubtedly  concerned,  as  well  as  red  carotinoid-like  pig- 
ments which  require  further  study. 

Carotinoids  have  been  identified  in  a  few  molds  and  yeasts  but 
their  nature  is  unknown. 

There  is  considerable  uncertainty  regarding  the  exact  relation  to 
the  carotinoids  of  certain  yellow  pigments  characterizing  the  slime 
molds,  but  xanthophyll  or  carotin-like  pigments  are  indicated  in  the 
case  of  certain  species. 

Carotinoids  are  formed  by  several  species  of  bacteria.  Carotin 
appears  to  be  the  principal  pigment  concerned  in  the  case  of  B. 
egregiwn  and  Spaerotilus  roseus.  The  exact  nature  of  the  pigment 
has  not  been  determined  in  the  case  of  the  other  species  in  which 
carotinoids  have  been  determined  to  be  present. 


124  CAROTINOIDS  AND  RELATED  PIGMENTS 

Carotinoid- forming  bacteria  afford  an  excellent  opportunity  for 
fixing  the  conditions  under  which  these  pigments  develop  and  thus 
throwing  some  light  on  the  true  functions  of  carotinoids  in  plants. 
The  growth  of  these  plants  is  subject  to  very  exact  laboratory  con- 
trol and  their  pigmentation  is  not  complicated  by  the  formation  of 
chlorophyll. 


Chapter  IV 
Carotinoids  in  the  Vertebrates 

It  is  by  no  means  a  new  idea  that  certain  pigments,  widely  dis- 
tributed among  animals,  resemble  closely  in  their  chemical  and  physi- 
cal properties,  as  well  as  in  color,  the  pigments  of  the  vegetable  king- 
dom which  were  considered  in  the  preceding  chapters.  This  point 
was  brought  out  in  Chapter  I.  The  demonstration  of  a  general  biologi- 
cal relationship  of  these  animal  pigments  to  the  plant  carotinoids  is, 
however,  comparatively  recent.  It  is  because  of  this  relationship 
that  one  is  justified  in  considering  the  carotinoids  of  plants  and  ani- 
mals in  one  treatise.  The  development  of  this  idea  and  the  experi- 
mental justification  for  it  are  reserved  for  presentation  in  a  later 
chapter.  It  is  accordingly  necessary  to  anticipate  this  discussion  at 
this  point  and  to  review  the  evidence  for  the  distribution  of  the  caro- 
tinoids among  animals  without  having  first  justified  the  basis  for 
this  distribution.  The  reader  is  therefore  asked  to  assume  for  the 
moment  that  the  yellow  to  orange-red  animal  pigments  which  have 
been  most  commonly  called  lipochromes  are  in  all  probability  true 
or  modified  plant  carotinoids.  For  certain  of  the  higher  animals 
proof  has  been  furnished  that  their  lipochromes  are  true  carotinoids, 
but  this  knowledge  does  not  as  yet  extend  very  far  down  the  scale 
of  animals.  However,  the  thread  is  picked  up  again  for  certain  of 
the  lower  animals  so  that  it  does  not  require  a  difficult  stretch  of 
imagination  to  fill  in  the  gap,  wide  as  it  is  indeed  admitted  to  be. 

Carotinoids  in  Mammals 

Corpus  luteum.  Bearing  in  mind  that  carotin  was  the  first  veg- 
etable chromolipoid  discovered,  it  is  an  interesting  fact  that  the  first 
mammalian  chromolipoid  to  be  isolated  in  crystalline  form  likewise 
eventually  proved  to  be  carotin.  The  pigment  referred  to  is  that  of 
the  corpus  luteum  of  the  cow,  first  described  by  Piccolo  and  Lieben 
(1866)  and  a  little  later,  apparently  independently,  by  Holm  (1867). 
As  already  mentioned  in  Chapter  I,  the  former  called  the  pigment 

125 


b 


126  CAROTINOIDS  AND  RELATED  PIGMENTS 

luteohamatoidin  or  haemolutein,  while  Holm  called  it  hamatoidin.  Of 
the  two  papers  mentioned  that  of  Holm,  only,  has  been  accessible  to 
the  writer.  It  is  gratifying  to  note  how  accurately  Holm  described 
the  crystalline  form,  the  color  of  the  crystals,  both  alone  and  when 
dissolved  in  various  solvents,  and  the  characteristic  blue  color  reac- 
tion with  nitric  acid,  all  of  which  later  helped  to  identify  the  pig- 
ment as  carotin.  The  close  relationship  of  the  corpus  luteum  pig- 
ment to  other  yellow  pigments  in  plants  and  animals  was  first  recog- 
nized by  Thudichum  (1869),  but  his  supposition  that  most  of  these 
pigments  were  identical  has  since  proved  to  be  without  foundation, 
although  his  ideas  in  this  respect  were  in  part  correct. 

Capranica  (1877)  likewise  isolated  the  corpus  luteum  pigment  from 
cow's  ovaries  and  obtained  it  in  crystalline  form.  The  general  prop- 
erties (color  reactions,  spectroscopic  absorption  bands  and  solubility) 
corresponded  so  closely  with  those  of  the  pigment  of  the  yolk  of  eggs 
(hen)  and  the  pigment  in  the  retina  of  the  eyes,  as  examined  by  this 
investigator,  that  he  regarded  the  three  pigments  as  identical.  This 
conclusion  led  him  to  regard  this  pigment  as  one  of  the  most  impor- 
tant substances  in  living  matter.  The  following  quotation  from 
Capranica's  paper  is,  to  say  the  least,  the  most  enthusiastic  concep- 
tion of  the  part  which  carotinoids  play  in  animal  life,  which  the  writer 
has  encountered.  "Diese  Substanz  muss  demgemass  als  eine  der 
phylogenetisch  altesten  chemischen  Verbindungen  des  thierischen 
Korpers  angesehen  werden.  Wir  diirfen  annehmen,  dass  schon  in  den 
ersten  Regungen  der  organischen  Materie  das  lichtempfindliche  Mole- 
cul  des  Lutein  vorhanden  sein.  Die  erste  Entstehung  dieses  Moleciils, 
kann  man  sich  denken,  war  das  'Fiat  Lux.'  Mit  ihr  begann  zwischen 
Sonne  und  organischer  Materie  jene  empfindende  Verbindung,  als 
deren  letzte  und  hochste  Frucht  wir  des  Menschen  sonnenhaftes  Auge 
anstaunen." 

The  full  significance  of  Capranica's  contributions,  however,  was  not 
appreciated  by  him  or  by  subsequent  investigators  of  animal  chromo- 
lipoids.  He  observed,  among  other  things,  that  petroleum  ether  and 
carbon  disulfide,  respectively,  would  quantitatively  remove  the  cor- 
pus luteum  pigment  from  its  alcoholic  solution.  The  development  of 
the  technic  for  separating  carotin  from  other  pigments  by  this  method 
is  a  comparatively  recent  achievement,  as  shown  in  the  preceding 
chapters.  If  Capranica  had  thought  to  apply  this  test  to  the  egg 
yolk  pigment  which  he  had  under  investigation  he  would  have  dis- 
covered a  difference  which  may  have  led  to  a  much  earlier  discovery 


CAROTINOIDS  IN  THE  VERTEBRATES  127 

of  the  true  relationship  of  the  corpus  luteum  and  egg  yolk  pigments 
to  each  other  and  to  other  similar  pigments  in  plants  and  animals. 
At  any  rate,  much  of  the  subsequent  confusion  of  different  pigments 
might,  perhaps,  have  been  avoided. 

Kiihne  (1878),  however,  was  forced  to  conclude  that  the  corpus 
luteum  and  egg  yolk  pigments  were  not  identical,  after  examining 
carefully  their  spectroscopic  absorption  properties.  No  further  study 
appears  to  have  been  made  of  the  corpus  luteum  pigment  until  Escher 
(1913)   definitely  established  its  identity  with  carotin. 

Before  referring  to  other  mammalian  carotinoids  it  may  be  well  to 
point  out  that  we  have  definite  proof  that  carotin  is  the  corpus  luteum 
pigment  only  in  the  case  of  cows  and  sheep,  from  which  Escher 
obtained  his  material  for  study.  Pigmented  tissue  appears  on  the 
human  ovary,  also,  but  there  is  no  evidence  that  the  pigment  is  exclu- 
sively carotin.  On  the  contrary  the  inference  which  may  be  drawn 
from  observations  regarding  the  character  of  the  chromolipoids  in 
other  parts  of  the  human  body  is  that  both  carotin  and  xanthophylls 
probably  appear  in  the  human  corpus  luteum.  Still  less  is  known 
regarding  the  pigment  in  the  corpus  luteum  of  other  mammals.  In 
the  horse  it  is  probably  carotin,  since  this  pigment  appears  in  the 
blood  of  that  animal.  Carotinoids  are  not  present  at  all  in  the  so- 
called  yellow  bodies  on  the  ovaries  of  swine,  as  pointed  out  by  van 
den  Bergh,  Muller  and  Broekmeyer  (1920).  The  writer  ^  succeeded  in 
extracting  a  small  amount  of  yellow  coloring  matter  from  swine 
ovaries  when  a  sufficient  number  were  extracted,  but  all  attempts  to 
identify  the  pigment  as  carotinoid  resulted  in  failure. 

Blood  serum.  Although  the  carotinoid  of  the  corpus  luteum  of  the 
cow  was  the  first  mammalian  chromolipoid  to  be  isolated  in  crystal- 
line form,  the  coloring  matter  in  the  blood  serum  of  cattle  was  prob- 
ably the  first  to  attract  attention.  Krukenberg  (1885a),  who  deserves 
credit  for  the  first  extensive  study  of  the  pigment,  mentions  the  much 
earlier  attempts  of  Samson  (1835),  Denis  (1838)  and  Schmidt  (1865) 
to  determine  its  nature.  It  is  true  that  Thudichum  (1869)  stated 
that  the  yellow  pigment  of  blood  serum  belonged  to  his  group  of 
luteins,  but  he  did  not  trouble  to  mention  the  animals  in  which  he 
had  found  it,  or  how  he  had  isolated  the  pigment.  As  a  matter  of  fact 
Krukenberg  (1885a)  found  it  to  be  rather  difficult  to  separate  the 
pigment  of  cattle  serum  from  the  other  blood  constituents;  direct 
extraction  with   all  the  known  fat  solvents  failed  completely,   and 

1  Unpublished  observations. 


128  CAROTINOIDS  AND  RELATED  PIGMENTS 

success  was  attained  only  by  repeated  extractions  of  the  serum  with 
amyl  alcohol.    The  writer's  study  of  blood  serum  pigments  of  cattle 
has  shown  that  this  difficulty  is  readily  explained.    When  the  chromo- 
lipoid  present  is  carotin,  the  pigment  is  physico-chemically  attached 
to  serum-albumin.    Alcohols  have  a  greater  attraction  than  pigment 
for  the  colloidal  protein  and  thus  replace  it.    Fat  solvents  will  then 
extract  the  pigment,  petroleum  ether  being  the  best  solvent  to  use. 
Krukenberg's  observations  of  the  pigment  isolated  by  him  from  ox 
serum  were  confined  to  solubility  properties  in  the  lipochrome  sol- 
vents, the  color  reactions  with  concentrated  HgSO^  and  HNO3,  and  . 
the  spectrum  bands  of  the  pigment.    Positive  identification  as  a  lipo- 
chrome was  secured  in  each  case.    Krukenberg  was  careful  to  recog- 
nize  that  pronounced   spectroscopic   differences   among   lipochromes 
indicated  the  existence  of  more  than  one  individual  in  his  lipochrome 
•  group.    On  these  grounds  he  was  led  to  conclude  that  the  blood  serum 
pigment  of  the  ox  is  probably  identical  with  the  lutein  of  the  corpus 
luteum,  whose  spectrum  properties  had  been  previously  pictured  by 
Kiihne.     The  additional  interesting  observation  was  made  that  the 
fresh  serum  itself  showed  the  spectrum  bands,  although  shifted  con- 
siderably towards  the  red  end  of  the  spectrum  from  their  position  in 
chloroform  or  ether.    The  writer  was  unable  to  verify  this  for  a  speci- 
men of  human   blood  serum  which  proved  to  be   rich   in  carotin. 
Although  Krukenberg  made  no  attempt  to  identify  the  pigment  with 
any  of  the  vegetable  lipochromes  with  which  he  was  familiar,  his 
graphic  representation  of  the  spectrum  of  the  cattle  serum  pigment 
shows  it  to  be  identical  with  that  of  carotin.     Krukenberg  had  no 
explanation  to  offer  for  the  occurrence  of  the  pigment  in  the  blood. 
He  was  opposed  to  the  view  that  it  originated  from  hemoglobin,  but 
nevertheless  saw  an  analogy  between  the  simultaneous  occurrence  of 
lipochrome  with  the  respiratory  pigment  of  both  plants  and  animals. 
Van  den  Bergh  and  Snapper  (1913)   confirmed  the  general  observa- 
tions of  Krukenberg  regarding  the  properties  of  the  pigment  of  cattle 
serum.    In  addition,  they  noted  traces  of  bilirubin  in  the  serum  and 
proposed  an  interesting  test  for  the  presence  of  both  lipochrome  and 
bilirubin  in  blood  serum  based  on  their  observation  that  the  lipo- 
chrome of  cattle  serum  is  precipitated  with  the  proteins  when  two 
volumes  of  95  per  cent  alcohol  are  added  to  one  volume  of  serum 
while  bilirubin  remains  in  the  supernatant  fluid  when  the  precipitated 
proteins  are  centrifugalized.    Definite  identification  of  the  lipochrome 
of  cattle  serum  as  carotin  was  made  by  Palmer  and  Eckles  (1914c) 


CAROTINOIDS  IN  THE  VERTEBRATES  129 

by  applying  the  numerous  macroscopic  tests  for  this  pigment  evolved 
by  the  plant  biochemists,  particularly  Willstatter  and  Mieg  and 
Tswett.  This  fact  has  recently  been  confirmed  by  van  den  Bergh 
and  Muller  (1920)  and  by  van  den  Bergh,  Muller  and  Broekmeyer 
(1920).  In  addition,  Palmer  and  Eckles  found  that  xanthophylls 
could  also  be  demonstrated  in  small  amounts  in  well-colored  serum  if 
sufficient  material  (250-300  c.c.)  was  used.  Neither  type  of  carotinoid 
was  present  in  the  blood  of  a  newborn  calf. 

After  Hammarsten  (1878)  isolated  crystalline  bilirubin  from  horse 
serum,  it  was  believed  for  many  years  that  this  pigment  was  the  sole 
cause  of  the  well-known  golden  yellow  color  of  the  serum  of  this  mam- 
mal. Gallerani  (1904),  however,  found  a  lipochrome-like  pigment 
accompanying  the  bilirubin  in  horse  serum,  for  which  he  proposed  the 
name  plasmachrome.  Van  den  Bergh  and  Snapper  (1913),  also, 
found  some  lipochrome  accompanying  the  bilirubin  in  horse  serum. 
The  carotinoid  identity  of  this  lipochrome  was  shown  a  little  later  by 
the  writer  (1916),  using  serum  from  a  horse  on  bluegrass  pasture 
(rich  in  carotinoids) .  Carotin  only  was  found,  adsorbed  on  the  albu- 
min, as  in  the  case  of  cattle  serum,  although  the  quantity  present  in 
a  unit  volume  was  considerably  less  than  was  found  in  cattle  serum 
under  comparable  feeding  conditions.  Van  den  Bergh  and  Muller 
and  Broekmeyer  (1920)  have  confirmed  these  findings,  also,  in  so  far 
as  the  character  of  the  carotinoid  and  the  amount  present  are 
concerned. 

Since  Thudichum's  (1869)  early  observation  it  has  been  recognized 
that  human  blood  serum  may  be  colored  by  a  lipochrome.  Zoja 
(1904)  found  that  bilirubin  is  not  present  except  under  pathological 
conditions.  However,  van  den  Bergh  and  Snapper  (1913)  state  that 
the  serum  of  normal  persons  always  contains  a  certain  amount  of 
both  lipochrome  and  bilirubin,  sometimes  one  and  sometimes  the  other 
being  in  excess.  They  observed,  also,  that  the  serum  of  diabetics  may 
contain  extraordinarily  large  amounts  of  lipochrome,  an  observation 
subsequently  confirmed  by  Umber  (1916),  Burger  and  Reinhart  (1918, 
1919),  Salomon  (1919),  van  den  Bergh  and  Muller  (1920),  van  den 
Bergh,  Muller  and  Broekmeyer  (1920),  and  by  Head  and  Johnson 
(1921).  Umber  was  able  to  shake  the  pigment  out  of  the  serum  with 
ether  alone.  Burger  and  Reinhart  (1918)  suggested  that  the  serum 
pigment  might  be  of  exogenous  origin  and  later  (1919)  presented 
quantitative  data  showing  a  rise  in  the  pigmentation  of  the  serum  on 
a  diet  of  green  food.     Salomon  definitely  identified  as  carotin  the 


130  CAROTINOIDS  AND  RELATED  PIGMENTS 

pigment  which  he  extracted  from  high  colored  human  serum.  Of 
interest  is  his  observation  that  in  this  case  direct  extraction  with 
ether  took  out  very  little  pigment,  it  being  necessary  first  to  precipi- 
tate the  proteins  with  alcohol  and  extract  the  precipitate  with  ether. 
Apparently  this  investigator  regarded  the  presence  of  the  carotin  as 
fortuitous,  for  he  mentions  the  difficulty  in  distinguishing  the  pig- 
ment from  the  normal  lipochrome  of  the  blood. 

It  is  obvious  that  none  of  the  workers  mentioned  in  the  preceding 
paragraph  were  familiar  with  the  observations  of  the  writer  on  the 
character  and  cause  of  the  normal  chromolipoid  of  cattle  and  horse 
serum.  It  remained  for  Hess  and  Myers  (1919)  to  show  the  direct 
application  of  the  writer's  observations  on  animals  to  the  variations 
in  the  pigmentation  of  human  blood  serum,  by  demonstrating  marked 
variations  in  the  carotin  content  of  the  blood  serum  of  children  with 
variations  in  the  carotin  content  of  their  diet.  These  observations 
have  been  extended  greatly  by  van  den  Bergh  and  Muller  (1920)  and 
van  den  Bergh,  Muller  and  Broekmeyer  (1920)  who  have  shown  that 
both  carotin  and  xanthophylls  play  a  part  in  causing  the  normal  pig- 
mentation of  human  blood  serum,  sometimes  one  and  sometimes  the 
other  predominating,  although  carotin  is  usually  in  excess. 

The  writer  has  recently  observed  an  interesting  case  of  marked 
change  in  the  character  of  the  carotinoid  in  the  blood  serum  of  an 
adult.  At  the  time  of  the  first  examination  the  serum  was  colored 
almost  exclusively  by  carotin,  which  could  not  be  shaken  out  of  the 
blood  with  ether.  At  this  time  carrots  played  a  large  part  in  the  diet. 
At  the  time  of  the  second  examination  the  pigment  was  readily  ex- 
tracted simply  by  shaking  the  serum  with  ether.  The  character  of 
the  diet  was  not  ascertained  in  this  case,  although  a  similar  pigment, 
readily  extracted  by  ether,  was  found  abundantly  in  the  blood  of  two 
other  persons  on  a  diet  rich  in  green  foods  (spinach  and  green  string 
beans).  By  analogy  with  the  writer's  (1915)  experiments  with  the 
pigment  of  fowl  serum  this  pigment  should  have  been  xanthophyll. 
However,  a  phase  test  applied  to  the  pigment  in  each  case  showed 
that  it  was  almost  quantitatively  epiphasic  between  the  petroleum 
ether  and  80  per  cent  methyl  alcohol.  Inasmuch  as  this  property  is 
supposed  to  be  distinctly  characteristic  of  carotin,  it  appears  that  the 
character  of  the  diet  may  influence  the  manner  in  which  carotin  is 
carried  by  human  blood.  In  each  case  the  serum  extracts  showed 
two-banded  absorption  spectra  when  using  a  spectroscope  with  nar- 
row dispersion,  but  it  was  not  possible  to  secure  the  measurements  of 


CAROTINOIDS  IN  THE  VERTEBRATES  131 

the  bands  when  using  an  instrument  of  high  dispersion  equipped  for 
measuring  the  wave-length  positions  of  the  bands. 

A  word  may  be  said  here  regarding  the  state  of  carotinoids  in 
blood.  Van  den  Bergh  and  Muller  (1920)  assert  that  neither  carotin 
nor  xanthophylls  can  be  shaken  out  of  blood  serum  with  ether.  They 
believe  that  the  pigments  are  always  in  colloidal  solution  in  the 
plasma.  The  writer  is  in  accord  with  this  view  in  so  far  as  carotin 
in  ox  and  horse  serum  is  concerned,  and  at  times  for  human  serum. 
It  is  believed,  however,  that  in  all  probability  a  double  colloidal 
phenomenon  is  involved  in  these  cases,  i.e.,  first,  a  colloidal  adsorption 
of  the  carotin  by  albumin  and  second,  a  colloidal  solution  of  this  albu- 
min in  the  plasma.  As  for  xanthophyll  in  blood  serum,  the  writer 
merely  wishes  to  state  that  he  has  never  failed  to  secure  its  direct 
extraction  with  ether  when  present  in  the  serum  of  animals,  and 
accordingly  does  not  feel  justified  in  believing  that  colloidal  phe- 
nomena are  involved  in  any  way.  The  explanation  for  this  differ- 
ence offers  an  interesting  problem  in  biochemistry. 

Observations  are  very  scanty  on  the  pigment  of  the  blood  serum 
of  other  mammals.  The  writer  (1916)  examined  the  blood  of  each  of 
three  breeds  of  swine,  representing  the  Duroc-Jersey,  Poland  China 
and  Berkshire  breeds  at  a  time  when  they  were  on  pasture,  but  failed 
to  detect  the  presence  of  even  traces  of  carotinoid  or  other  chromo- 
lipoid-like  pigment.  In  a  similar  manner  the  blood  of  each  of  five 
breeds  of  sheep,  namely,  Dorset,  Hampshire,  Merino,  Shropshire  and 
Southdown,  showed  the  presence  of  only  traces  of  chromolipoid,  which 
appeared  to  be  carotin,  although  the  animals,  like  the  swine,  were 
receiving  an  abundance  of  carotinoid-rich  pasture  grass  at  the  time. 
The  blood  of  an  Angora  goat,  under  like  feeding  conditions,  showed 
traces  of  carotinoid  also.  Van  den  Bergh,.  Muller  and  Broekmeyer 
(1920)  likewise  found  no  carotinoids  in  the  blood  serum  of  swine, 
guinea  pigs  or  dogs,  and  traces  only  in  the  blood  serum  of  cats.  In 
the  case  of  the  latter  animal,  xanthophyll  practically  disappeared 
from  the  blood  within  a  half  hour  after  an  intravenous  injection  of  a 
colloidal  solution  of  xanthophyll.  It  is  stated  that  the  pigment  was 
found,  however,  in  the  liver. 

Milk  fat.  Thudichum's  classic  paper  included  the  pigment  of 
butter  fat  among  the  "luteins."  Blythe  (1879),  however,  regarded 
the  alcohol  soluble  lactochrome  which  he  isolated  from  milk  whey  as 
the  cause  of  the  butter  fat  color,  and  Desmouliere  and  Gautrelet 
(1903)  concluded,  after  isolating  a  urobilin-like  pigment  from  milk, 


132  CAROTlNOim  AND  RELATED  PIGMENTS 

that  no  lipochromes  are  present.  On  the  other  hand  the  fact  that  the 
pigment  of  butter  fat  appears  in  the  unsaponifiable  ether  extractable 
material  at  once  classifies  it  as  a  chromolipoid.  Palmer  and  Eckles 
(1914a)  were  the  first  to  make  a  critical  examination  of  the  pigment 
from  the  standpoint  of  the  plant  carotinoids,  finding,  as  might  be 
expected  in  the  light  of  Escher's  work  on  the  corpus  luteum  pigment 
of  the  cow,  that  the  pigment  corresponds  exactly  in  physical  and 
chemical  properties  (spectroscopic,  solubility  and  phase  test)  with 
carotin.  In  addition  we  found,  when  the  phase  test  and  a  chroma- 
tographic analysis  were  applied,  that  small  amounts  of  xanthophylls 
usually  accompany  the  carotin.  These  were  most  evident  in  highly 
colored  butter  fat,  a  chromatogram  in  one  case  showing  two  and  pos- 
sibly three  distinct  adsorption  zones  of  xanthophyll.  The  pigment 
in  each  of  these  zones  showed  the  xanthophyll  absorption  bands  and 
were  hypophasic  in  the  phase  test  between  petroleum  ether  and  80 
per  cent  alcohol. 

The  character  of  the  carotinoids  in  the  milk  fat  of  other  animals 
has  not  been  determined.  Palmer  (1916)  and  Palmer  and  Kennedy 
(1921)  have  noted  the  presence  of  carotinoid  in  traces  in  the  milk  fat 
of  sheep  and  goats  without  determining  which  kind  of  carotinoid  is 
present.  We  have  also  noted  a  complete  absence  of  carotinoids  from 
the  milk  fat  of  albino  rats  and  swine,  even  the  fat  of  the  colostrum 
milk  of  the  latter. 

The  fat  of  human  milk  is  always  more  or  less  pigmented,  that  of 
colostrum  being  especially  highly  pigmented.  Palmer  and  Eckles 
(1914e)  found  both  carotin  and  xanthophylls  in  about  equal  quan- 
tities, as  judged  from  the  color  of  the  solutions  obtained  in  the  phase 
test  when  applied  to  the  isolated  pigment.  Two  samples  of  human 
milk  were  examined,  from  different  individuals,  one  sample  being 
colostrum.  This  result  is  to  be  expected  in  the  light  of  what  has  been 
found  subsequently  regarding  the  presence  of  both  types  of  carotinoids 
in  human  blood. 

Adipose  tissue.  The  adipose  tissue  of  cattle,  horses  and  man  is 
characterized  by  varying  amount  of  pigment,  which  at  times  attains  a 
high  concentration  in  the  horse,  in  certain  breeds  of  cattle,  such  as 
the  Jersey  and  Guernsey  dairy  breeds,  and  at  times  in  man.  The 
adipose  tissue  of  other  species  of  mammals,  including  sheep  and  goats, 
dogs,  cats,  rabbits,  swine,  rats,  guinea  pigs  and  other  rodents,  is 
entirely  or  almost  entirely  devoid  of  pigment.  In  the  cases  of  pig- 
mented adipose  tissue  of  cattle  Palmer  and  Eckles  (1914b)  found  the 


CAROTINOIDS  IN  THE  VERTEBRATES  133 

pigment  to  be  chiefly  carotin,  with  some  admixed  xanthophylls.  In 
the  case  of  the  adipose  tissue  of  the  horse  van  den  Bergh,  Muller  and 
Broekmeyer  (1920)  found  carotin  exclusively.  The  latter  investigators 
have  made  the  only  examination  of  human  adipose  tissue.  Varying 
amounts  of  pigment  and  varying  proportions  of  carotin  and  xantho- 
phyll  were  found  in  numerous  specimens  obtained  on  autopsy  of  indi- 
viduals dead  of  various  disorders.  In  most  cases  carotin  was  somewhat 
in  excess  of  xanthophyll.  Of  interest  in  this  connection  is  the  obser- 
vation of  Krukenberg  and  Wagner  (1885)  of  a  yellow  lipochrome  in 
human  bone  marrow.  The  position  of  the  spectroscopic  absorption 
bands  of  the  pigment  which  are  shown  in  a  drawing  by  these  authors 
resembles  xanthophyll  rather  than  carotin  inasmuch  as  the  maximum 
absorption  of  the  first  band  is  on  the  violet  side  of  the  F  line,  while 
the  maximum  absorption  of  carotin,  as  we  now  know,  is  at  the  F  line. 

Internal  organs.  As  van  den  Bergh,  Muller  and  Broekmeyer  (1920) 
have  shown  in  their  extensive  study  of  carotinoids  in  the  human  and 
animal  body,  certain  of  the  internal  organs  of  mammals  appear  to  have 
an  elective  affinity  for  carotinoids  which  is  greater  than  can  be  ex- 
plained by  their  fat  content.  Krukenberg  (1885b)  first  called  atten- 
tion to  the  presence  of  lipochrome  in  human  and  animal  adrenals,  at 
times  in  high  concentration  in  the  human  glands.  He  described  its 
extraction  with  hot  alcohol,  its  absorption  bands  resembling  those  of 
the  lipochrome  of  cattle  serum,  and  the  color  reactions  with  con. 
H2SO4  and  HNO3.  This  was  confirmed  for  the  human  adrenals  by 
Lubarsch  (1902),  Sehrt  (1904)  and  Hueck  (1912).  Sehrt  concluded 
that  the  lipochrome  was  different  from  the  plant  lipochromes  (caro- 
tinoids). Findlay  (1920)  and  also  van  den  Bergh,  Muller  and  Broek- 
meyer (1920)  have  examined  the  pigment  of  human  adrenals  from  the 
standpoint  of  carotinoid  properties.  Both  carotin  and  xanthophylls 
were  demonstrated,  the  latter  authors  reporting  data  for  a  large  number 
of  cases.  The  technic  of  Findlay  is  to  be  criticized,  however,  in  that 
he  drew  his  conclusion  regarding  both  types  of  carotinoids  being 
present  by  applying  the  phase  test  for  the  carotinoids  directly  to  the 
issues.  In  other  words,  he  regarded  pigments  which  did  not  readily 
dissolve  out  of  the  tissue  with  petroleum  ether  as  xanthophyll,  while 
that  which  was  extracted  by  this  solvent  he  regarded  as  carotin.  It 
is  doubtful  whether  the  phase  test  can  be  applied  except  to  solutions 
of  the  carotinoids. 

Van  den  Bergh,  Muller  and  Broekmeyer  examined  the  suprarenals 
of  the  horse,  guinea  pig,  cat,  dog,  and  swine  for  carotinoids.     They 


134  CAROTINOIDS  AND  RELATED  PIGMENTS 

report  carotin  in  all  cases,  the  amount  varying  from  relatively  large 
amounts  in  the  case  of  the  horse  and  guinea  pig  to  very  little  in  the 
case  of  swine,  cats  and  dogs.  Findlay  reported  a  small  amount  of 
carotin-like  pigment  in  the  suprarenals  of  the  sheep.  Palmer  and 
Kennedy  (1921)  were  unable  to  find  any  pigment  soluble  in  alcohol 
or  petroleum  ether  in  the  suprarenals  of  albino  rats. 

There  has  been  very  little  study  of  the  pigments  of  mammalian 
liver  from  the  standpoint  of  carotinoids.  It  is  difficult  tissue  to 
examine  because  it  is  rich  in  pigments  of  unknown  character  which 
are  soluble  in  certain  of  the  fat  solvents,  and  also  because  one  of 
these  pigments,  at  least,  gives  a  color  reaction  with  con.  H^SO^ 
which  may  easily  be  mistaken  for  a  carotinoid  reaction.  It  is  to  be 
expected  that  the  liver  of  animals  whose  blood  and  adipose  tissue 
may  be  rich  in  carotinoids  will  also  contain  these  pigments,  e.g.,  that 
the  human  liver  will  contain  varying  amounts  of  both  carotin  and 
xanthophylls,  and  that  the  liver  of  the  cow  and  horse  will  contain 
carotin.  Van  den  Bergh,  Muller  and  Broekmeyer  (1920)  have  found 
this  to  be  the  case.  On  the  other  hand  the  statement  of  these  investi- 
gators that  the  liver  of  swine,  cats,  dogs  and  guinea  pigs  contains 
small  amounts  of  carotinoids  is  to  be  accepted  with  reserve  until  the 
experimental  evidence  for  this  statement  is  extended  to  include  the 
spectroscopic  and  adsorption  properties  of  the  pigments  isolated. 
These  investigators  based  their  conclusions  on  color  reactions  with 
concentrated  acids  and  upon  solubility  in  carotinoid  solvents  and  upon 
the  phase  test.  None  of  these  properties  is  properly  to  be  regarded 
as  specific  for  carotinoids. 

Nerves.  Meschede  (1865,  1872)  first  observed  yellow  pigment  in 
nerve  cells  which  could  be  extracted  with  fat  solvents.  Rosin  (1896) 
first  associated  the  pigment  with  the  lipochromes  then  rising  into 
prominence.  He  noted  its  presence  in  the  human  and  in  cattle,  and 
its  absence  from  the  nerve  cells  of  the  dog,  cat,  rabbit,  rat  and  mouse. 
Rosin  and  Fenyvessey  (1900)  noted  that  the  pigment  was  absent  from 
the  nerve  cells  of  the  new  born,  but  that  it  was  always  present  in 
the  nerve  cell  tissue  of  adult  humans.  Following  the  studies  of  Lu- 
barsch  (1902),  who  regarded  the  lipochrome  as  an  ''abnutzung" 
(wear-and-tear)  product  of  endogenous  origin,  pathologists  have  at- 
tached significance  to  the  increase  in  lipochrome  pigmentation  in  nerve 
cells  which  have  been  observed  in  disease.  Dplley  and  Guthrie  (1919), 
however,  have  made  a  careful  study  of  the  occurrence  of  chromolipoid 
in  the  nerve  cell  of  man  and  animals  and  have  found  that  it  can  be 


CAROTINOIDS  IN  THE  VERTEBRATES  135 

demonstrated  microchemically  only  in  those  species  of  animals  in 
which  carotinoids  normally  occur,  e.g.,  man,  cow  and  fowl.  The  con- 
clusion which  they  drew  seems  incontrovertible,  namely,  that  the 
chromolipoids  of  these  tissues  are  true  carotinoids  of  exogenous  origin, 
the  type  of  pigment  being  governed  by  the  species  of  animal,  i.e.,  by 
the  type  of  carotinoid  resorbed. 

Lipochrome  pigment  is  also  present  in  other  body  tissue  of  man, 
i.e.,  in  the  seminal  vesicle  (Maass,  1889)  and  in  the  epithelial  muscle 
cells  (Akutsu,  1902)  and  in  the  heart,  where  Dolley  and  Guthrie 
(1921)  have  shown  its  carotinoid  nature.  Of  interest  is  Akutsu's  ob- 
servation that  the  pigment  is  absent  from  these  tissues  in  the  case  of 
new  born  babies  and  young  children,  and  begins  to  occur  about  the 
age  of  puberty. 

Skin.  The  skin  of  dairy  cattle,  especially  that  of  the  Jersey  and 
Guernsey  breeds,  is  often  characterized  by  a  high  yellow  color,  which 
is  often  almost  orange  in  hue.  The  wax  in  the  ears  of  these  animals 
is  also  highly  pigmented.  The  skin  color  is  especially  noticeable  on 
the  udder,  particularly  the  escutcheon.  Using  the  ear  wax  as  the 
source  of  material.  Palmer  and  Eckles  (1914b)  found  the  pigment  to 
be  carotin,  chiefly,  with  a  little  xanthophyll. 

Smith  (1893)  observed  a  yellow  pigment  in  the  "dandruff"  of  the 
horse,  which  he  regarded  as  modified  chlorophyll.  Inasmuch  as  he 
observed  a  variation  in  the  amount  of  this  pigment  with  the  food  of 
the  animal  the  conclusion  seems  obvious  that  the  pigment  was  carotin, 
which  characterizes  the  blood  serum  and  adipose  tissue  of  this  species. 

Carotinoids  may  also  color  the  human  skin.  Moro  (1908),  Kaup 
(1919),  Stollzner  (1919),  Klose  (1919)  and  Hess  and  Myers  (1919) 
noted  skin  coloration  in  children  after  eating  heavily  of  carrots.  Most 
of  these  writers  associated  the  coloration  with  the  carotin  in  the 
carrots,  but  all  of  them,  except  Hess  and  Myers,  regarded  the  phe- 
nomenon as  an  abnormality.  The  latter,  only,  pictured  the  phenomenon 
as  an  exaggeration  of  a  normal  condition  and  demonstrated  the  carotin 
in  the  blood  serum.  They  state  that  the  feeding  of  oranges  or  eggs 
to  children  may  result  in  a  similar  skin  coloration.  Schiissler  (1919) 
and  Salomon  (1919)  have  noted  similar  phenomena  in  adults  -  the 
entire  body  being  affected  in  the  cases  cited  by  ScLiissler.    These  were 

"  Hashimato  (1922)  states  that  a  yellow  skin  pigmentation  of  dietary  origin  was 
described  in  the  Japanese  literature  by  Baelz  as  early  as  1896  and  called,  "aurantia- 
sis  cutis."  It  is  also  pointed  out  that  Miura  (1917),  a  Japanese  writer,  ascribed  the 
pigmentation  to  carotin  and  used  the  term,  "carotinosis."  Hashimato  reports  35 
cases  among  adults  as  the  result  of  excessive  eating  of  squash. 


136  CAROTINOWS  AND  RELATED  PIGMENTS 

due  to  a  carrot  diet.  Carotin  was  inferred,  not  demonstrated,  in  these 
cases,  although  Salomon  measured  the  extent  of  the  "xanthemia"  in 
certain  individuals  by  determining  the  extinction  coefficient  of  the 
absorption  bands  of  the  ether  extract  of  the  blood. 

Von  Noorden  (1904)  first  called  attention  to  a  frequent  yellow  skin 
coloration  in  diabetics,  which  was  not  due  to  jaundice.  He  proposed 
for  it  the  name  Xanthosis  Diabetica.  Van  den  Bergh  and  Snapper 
(1913)  also  called  attention  to  the  phenomenon  and  showed  in  addition 
that  it  was  accompanied  by  an  increased  lipochrome  content  of  the 
blood  serum,  which  they  regarded  as  the  cause.  Umber  (1916)  noticed 
the  same  correlation  in  cases  of  Xanthosis  Diabetica.  Burger  and 
Reinhart  (1918)  first  suggested  an  exogenous  origin  of  the  pathological 
phenomenon,  for  which  they  later  (1910),  as  well  as  Salomon  (1919), 
offered  proof.  Hess  and  Myers  (1919)  saw  the  correlation  between 
the  pathological  and  normal  skin  colorations  on  carotinoid  rich  diets, 
and  van  den  Bergh  and  Muller  (1920)  and  van  den  Bergh,  Muller  and 
Broekmeyer  (1920)  have  presented  such  extensive  data  on  the  pres- 
ence of  carotinoids  in  the  human  organism  that  their  conclusion  seems 
entirely  justified  that  the  skin  colorations  of  diabetics  is  due  pri- 
marily to  the  vegetarian  character  of  the  diet  of  persons  afflicted  with 
this  disease.  It  is  not  to  be  inferred,  however,  that  carotin  is  always 
the  cause  of  the  skin  coloration.  Head  and  Johnson  (1921)  with  the 
assistance  of  the  writer  have  demonstrated  carotin  as  the  sole  cause 
of  one  case  where  the  diet  of  the  diabetic  was  rich  in  carotin  (the 
patient  ate  heavily  of  carrots),  the  skin  clearing  up  when  the  source 
was  removed.  On  the  other  hand  another  case  of  skin  coloration  of 
a  diabetic  has  come  under  the  observation  of  the  writer  which  was 
evidently  due  largely,  is  not  entirely,  to  xanthophylls.  The  diet  was 
rich  in  xanthophylls  (eggs  and  green  beans),  and  the  blood  serum 
showed  much  xanthophyll  with  little  carotin.  The  skin  in  this  case 
w^as  also  cleared  up  by  removing  the  source  of  the  pigment. 

In  view  of  the  fact  that  carotinoids  have  been  found  in  the  skin  of 
both  normal  and  diseased  persons,  it  seems  doubtful  whether  any 
pathological  significance  can  be  attached  to  its  appearance  in  the  skin. 
Van  den  Bergh,  Muller  and  Broekmeyer  (1920),  who  have  studied 
this  question  extensively,  were  unable  to  note  any  correlations  between 
the  pigmentation  of  various  tissues  and  the  character  of  the  disease. 
The  difiiculty,  of  course,  is  that  the  pigments  must  be  of  dietary  origin. 
Even  a  diabetic  could  not  show  a  xanthosis  unless  his  diet  contained 
carotinoids.    On  the  other  hand  the  more  frequent  observation  of  an 


CAROTINOIDS  IN  THE  VERTEBRATES  137 

epidermal  xanthosis  in  diabetics  than  in  well  persons,  and  the  fact 
that  a  yellow  color,  presumably  of  the  same  origin,  is  frequently  seen 
in  the  palms  of  the  hands  and  on  the  soles  of  the  feet  of  persons  with 
acute  sickness,^  may  have  a  secondary  origin.  The  normal  cause  of 
the  disappearance  of  carotinoids  from  both  plants  and  animals  is  an 
oxidation.  This  is  undoubtedly  their  ultimate  fate  in  animals  unless 
they  are  secreted  in  the  milk  fat  or  egg  yolk  (in  fowls)  or  stored  up 
as  adipose  tissue  and  thus  protected  from  oxidation.  Where  the  oxida- 
tive tone  of  the  body  is  low,  as  in  diabetes,  coupled  in  many  cases 
with  abnormally  large  intake  of  carotinoids,  it  is  not  surprising  that 
the  pigments  should  appear  in  the  tissues  in  abnormally  large  amounts. 
This  is  especially  likely  to  be  true  of  the  epidermal  tissues  inasmuch 
as  the  effect  of  eating  carotinoid-rich  diets  in  normal  persons  shows 
that  the  subcutaneous  glands  can  serve  as  an  excretory  medium  for 
these  pigments. 

Carotinoids  in  Birds 

The  chromolipoid  pigments  of  birds  offer  many  of  the  most  inter- 
esting problems  in  the  field  of  animal  chromatology.  This  is  true  in 
spite  of  the  fact  that  a  cursory  knowledge  of  the  present  status  of  the 
question  of  carotinoid  pigmentation  in  the  case  of  the  domestic  fowl 
would  lead  one  to  believe  that  the  character  of  the  carotinoid  pig- 
ments found  in  the  feathered  animals,  as  well  as  the  origin  of  the 
pigments,  has  been  settled  for  all  species  of  birds.  This  belief  is  not 
justified.  Who  knows,  for  example,  whether  or  not  numerous  species 
lack  carotinoids  entirely,  as  is  the  case,  or  nearly  so,  with  many 
domestic  mammals?  This  is  a  relatively  simple  problem  to  solve. 
But  what  shall  one  say  of  the  problem  of  determining  why  the  type 
of  carotinoid  in  the  hen  is  different  from  that  of  the  cow;  or  of  the 
problem  of  ascertaining  why  the  xanthophyll  of  the  yolk  of  the  hen's 
egg  appears  to  be  chemically  an  isomer  of  plant  xanthophyll  in  spite 
of  the  fact  that  the  plant  xanthophyll  is  the  source  from  which  the 
hen  derives  the  pigment  for  the  egg  yolk ;  or  of  the  problem  of  explain- 
ing the  wide  variation  in  the  appearance  of  carotinoid  in  the  epidermis 
of  fowls,  in  all  of  which  the  adipose  tissue  is  highly  colored  with 
xanthophyll,  as  well  as  the  egg  yolk?  What  might  be  expected  to  be 
simply  physiological  problems  in  connection  with  the  behavior  of 
carotinoids  in  the  animal  organism  turn  out  to  be  complicated,  or  at 

'  Van  den  Bergh,  Muller  and  Broekmeyer  (1920)  state  that  this  phenomenon  has  been 
described  for  a  long  time  by  French  physicians  under  the  name  "signe  palmaire." 


138  CAROTINOIDS  AND  RELATED  PIGMENTS 

least  baffling.  For  example,  it  would  not  seem  unlikely  that  xantho- 
phyll,  readily  soluble  in  fat,  would  act  like  a  fat  dye  in  the  body  of 
the  hen.  However,  when  Palmer  and  Kempster  (1919b,  c)  fed  Sudan 
III  to  a  carotinoid-free  cockerel,  the  dye  quickly  appeared  in  the 
adipose  tissue  and  bone  marrow,  but  not  in  the  visible  skin  parts 
(shanks,  beak,  ear  lobes,  etc.),  whereas  xanthophyll,  when  fed  to  a 
carotinoid-free  cockerel  of  the  same  breed  appeared  in  the  shank  skin 
within  72  hours,  and  annatto,  a  different  fat  dye,  did  not  appear  in  the 
body  at  all.  Again,  when  Sudan  III  was  fed  to  a  laying  carotinoid- 
free  hen,  the  dye  quickly  appeared  in  the  egg  yolks  and  deeply  stained 
the  adipose  tissue,  whereas  xanthophyll,  when  fed  to  a  carotinoid-free 
laying  hen  appeared  only  in  the  egg  yolk,  the  adipose  tissue  and  epi- 
dermis being  unaffected  even  after  a  month  of  xanthophyll  feeding. 
How  are  these  interesting  observations  to  be  explained? 

Egg  yolk.    It  is  to  be  expected  that  the  pigment  of  the  yolk  of 
hen's  eggs  should  be  the  first  of  the  bird  chromolipoids  to  attract  the 
attention  of  the  physiologists.    Stadeler  (1867)  was  the  first  to  attempt 
to  secure  crystals  of  the  pigment.    He  failed  to  do  so,  but  observed 
the  solubility  of  the  pigment  in  ether  and  chloroform  with  a  golden 
yellow  color,  in  CS2  with  an  orange  color,  its  unsaponifiability,  and 
the  fact  that  HNO3,  containing  NO2,  imparted  a  dirty  blue-green 
color  to  the  impure  pigment,  while  a  trace  of  con.  H2SO4  had  the  same 
effect.     Thudichum   (1869),  as  already  mentioned,  included  the  pig- 
ment among  his  luteins.     Capranica  (1877)  mentioned  having  noticed 
the  similarity  in  properties  of  the  pigment  with  that  of  the  corpus 
luteum.    The  first  detailed  description  of  the  spectroscopic  absorption 
properties  of  the   egg  yolk  pigment  was  given  by  Kiihne    (1878). 
Careful  drawings  of  the  pigment  spectrum  in  ether,  petroleum  ether 
and  CS2  in  comparison  with  a  similar  spectrum  of  the  corpus  luteum 
pigment,  show  differences  now  readily  explained  in  the  light  of  our 
knowledge  regarding  the  type  of  carotinoid  involved  in  each  case. 
Kiihne,  as  already  mentioned,  decided  against  an  identify  of  the  two 
pigments  on  spectroscopic  grounds  and  also  because  the  egg  yolk  pig- 
ment failed  to  give  the  blue  color  reaction  with  iodine  previously  noted 
for  the  corpus  luteum  pigment.     Of  interest  is  Kiihne's  observation 
that  the  egg  yolk  pigment  is  soluble  in  bile.  '  Palmer  and  Eckles 
(1914d)  have  attached  some  significance  to  the  fact  that  plant  xantho- 
phyll is  soluble  in  bile  (ox)  while  carotin  is  not,  as  a  possible  explana- 
tion of  some  of  the  physiological  differences  between  these  types  of 
carotinoids  in  the  animal  body. 


CAROTINOIDS  IN  THE  VERTEBRATES  139 

The  probability  of  a  definite  chemical  relation  between  the  egg  yolk 
pigment  and  plant  carotinoids  was  pointed  out  for  the  first  time  by 
Schunck  (1903)  who  found  that  the  spectrum  of  the  alcoholic  solution 
was  identical  with  one  of  the  xanthophyll  group  of  pigments  which 
he  isolated  from  a  number  of  flowers.  Schunck's  work  is  described  in 
Chapter  II.  It  was  pointed  out  there  that  Schunck's  method  for  sep- 
arating the  xanthophylls  is  not  exact.  In  all  probability,  however, 
the  L  xanthophyll  which  he  described,  and  which  showed  the  same 
spectroscopic  properties  as  the  egg  yolk  lipochrome,  corresponds  best 
with  Tswett's  a  xanthophyll.  This  appears  to  be  the  xanthophyll 
which  is  present  in  the  chloroplastids  in  greatest  amount. 

The  definite  chemical  identification  of  the  egg  yolk  pigment  of  hen's 
eggs  as  xanthophyll  soon  followed,  when  Willstatter  and  Escher  (1912) 
isolated  the  crystalline  pigment  and  showed  that  it  corresponds  in 
all  its  chemical  and  physical  properties,  except  its  melting  point,  with 
the  crystaUine  xanthophyll  of  green  plants.  The  failure  of  the  egg 
yolk  xanthophyll  to  correspond  in  its  melting  point  with  the  plant 
xanthophyll  of  Willstatter  and  Mieg  (1907)  has  never  been  explained. 
Serono' (1912)  has  criticized  Willstatter  and  Escher's  work  severely, 
and  expressed  the  opinion  that  the  product  which  they  isolated  was 
not  a  carotinoid  at  all,  but  a  cholesterol  ester  of  oleic  acid.  He  shows 
how  the  elementary  composition  of  such  an  ester  corresponds  even 
more  closely  with  the  analyses  of  the  egg  yolk  xanthophyll  than  the 
latter  does  with  plant  xanthophyll,  and  advances  the  belief  that  this 
explains  the  high  melting  point  found  by  Willstatter  and  Escher  for 
the  egg  yolk  pigment.  Serono's  explanation  of  the  high  melting  point 
of  the  egg  yolk  xanthophyll  falls  to  the  ground,  however,  in  the  light 
of  the  studies  of  the  writer  (1915)  which  showed  that  the  egg  yolk 
pigment  is  not  only  chemically  related  to  plant  xanthophyll  but  is 
biologically  derived  from  it.  Whether  the  hen's  body  modifies  slightly 
the  plant  pigment,  thus  giving  it  a  different  melting  point  from  its 
precursor,  or  whether  the  hen  selects  one  of  the  several  plant  xantho- 
phylls differing  in  melting  point  from  the  mixed  product  obtained  by 
Willstatter  and  Mieg  from  nettle  leaves,  or  whether  the  difference  is 
to  be  explained  on  other  grounds  cannot  be  decided  definitely  at  the 
present  time, 

Xanthophyll  is  not  the  only  carotinoid  in  the  yolk  of  hen's  eggs. 
In  their  isolation  of  the  crystalline  pigment  Willstatter  and  Escher 
noticed  the  presence  of  a  small  amount  of  pigment  with  the  solubility 
relations  of  carotin.    The  writer  (1915)  was  able  to  confirm  this  in  his 


140  CAROTINOIDS  AND  RELATED  PIGMENTS 

study  of  the  biological  origin  of  the  egg  yolk  xanthophyll.  In  Will- 
statter  and  Escher's  study,  however,  the  bulk  of  their  carotin-like  pig- 
ment was  saponifiable,  so  that  its  actual  identity  with  carotin  remains 
doubtful. 

Xanthophyll  pigmented  egg  yolks  may  not  be  normal  for  all  species 
of  birds.  Krukenberg  (1882m)  examined  the  yolk  of  the  eggs  from 
two  breeds  of  parrots.  The  yolk  was  colorless  in  one  case,  but  the 
other  was  weakly  tinted  with  a  pigment  whose  spectrum  is  unques- 
tionably that  of  xanthophyll. 

Body  tissues.  It  is  not  to  be  expected  that  animals  whose  eggs  are 
highly  colored  with  carotinoid  should  be  devoid  of  the  pigment  in 
their  body  tissues.  Halliburton  (1886)  showed  that  the  blood  serum 
and  adipose  tissue  of  the  hen,  pigeon  and  dove  contains  lipochrome, 
but  the  descriptions  given  do  not  make  it  possible  to  decide  the  char- 
acter of  the  carotinoid  involved.  The  writer  has  observed  that  the 
pigment  in  pigeon  serum  may  be  extracted  by  shaking  with  ether, 
which  fact  may  indicate  its  xanthophyll  nature.  Schunck's  (1903) 
spectroscopic  studies  included  the  pigment  of  the  hen's  blood  serum. 
The  same  xanthophyll  was  found  as  in  the  egg  yolk.  The  writer's 
(1915)  study  of  the  fowl's  blood  also  showed  xanthophyll  to  be  the 
major  pigment  present  in  the  serum,  directly  extractable  with  ether  in 
all  cases  in  his  work. 

Krukenberg's  (1882b)  spectroscopic  drawings  of  the  yellow  skin  pig- 
ments of  pigeons,  hens  and  geese  resemble  very  closely  the  known 
spectra  for  xanthophyll.  We  now  know  that  this  pigment  is  xantho- 
phyll, at  least  in  the  case  of  fowls,  the  extracts  showing  the  phase  test 
and  spectroscopic  properties  of  this  pigment.  Van  den  Bergh  and 
Muller  (1920)  and  van  den  Bergh,  Muller  and  Broekmeyer  (1920) 
have  confirmed  these  observations  with  the  exception  of  the  direct 
extraction  of  the  xanthophyll  from  fowl  serum  by  ether.  In  only  two 
out  of  13  cases  were  they  able  to  shake  the  pigment  out  with  ether. 
The  explanation  of  this  divergence  in  their  observations  from  those 
made  by  the  writer  is  not  at  present  apparent. 

Retina.  It  has  been  known  since  the  early  observation  of  Hannover 
(1840)  that  globules  varying  in  color  from  red  to  greenish-yellow  occur 
in  the  retina  of  the  eyes  of  many  animals  and  birds.  The  physiolo- 
gists were  greatly  interested  in  these  pigmented  globules  during  the 
latter  half  of  the  19th  century,  pai-ticularly  as  to  the  possibility  of 
their  being  related  to  the  so-called  visual  pigments  of  the  eye.  These 
colored  globules  interest  us,  however,  only  in  so  far  as  the  character 


CAROTIN OIDS  IN  THE  VERTEBRATES  141 

of  the  pigments  is  concerned.  Unfortunately  no  modern  investigation 
of  these  pigments  has  been  made,  so  that  it  is  necessary  to  rely  on 
the  observations  of  those  who  were  unfamiliar  with  the  possibility  of 
their  being  related  to  plant  carotinoids.  The  blue  color  reaction  of 
the  lipochromes  with  iodine  was  introduced  by  Schwalbe  (1874)  in 
connection  with  these  retinal  pigments.  The  splendid  early  work  of 
Capranica  (1877)  on  the  chromolipoids  of  the  corpus  luteum  and  egg 
yolk  was  undertaken  primarily  to  study  the  yellow  to  red  retinal  pig- 
ments of  amphibians  and  birds.  He  found  a  complete  correspondence 
between  the  retinal  pigments  of  birds  and  those  of  the  egg  yolk. 

Kiihne  contributed  several  papers  on  the  retinal  pigments,  which 
appeared  in  the  memoirs  of  the  Physiological  Institute  of  the  Uni- 
versity of  Heidelberg.  Reference  has  already  been  made  to  the  only 
one  of  these  papers  which  has  been  accessible  to  the  writer  (Kiihne, 
1878),  which  is  presumably  the  only  paper  reporting  Kiihne's  study 
of  the  chemical  and  physical  properties  of  the  pigments.  According 
to  this  investigator  the  microscope  reveals  oil  globules  of  three  colors 
in  the  retinal  epithelium  of  fowls,  namely  red,  yellow  and  greenish- 
yellow.  Kiihne's  study  of  these  globules  led  him  to  conclude  that 
three  distinct  pigments  were  involved,  which  he  called  rhodophane, 
xanthophane  and  chlorophane,  respectively.  The  evidence  for  the 
existence  of  three  pigments  was  based  on  the  observations:  (1)  that 
when  a  dry  sodium  soap  was  prepared  of  the  orange-red  ether  extract 
of  the  retinas  and  submitted  to  successive  extractions  with  petroleum 
ether,  ether  and  benzene  until  each  solvent  extracted  no  more  pigment, 
the  extracts  were,  in  succession,  yellowish-green,  orange  and  rose-red 
in  color;  (2)  the  chlorophane  in  the  yellowish-green  petroleum  ether 
could  be  purified  from  admixed  xanthophane  by  repeated  evaporations 
and  extractions  with  petroleum  ether,  giving  solutions  more  and  more 
green  in  color;  (3)  the  xanthophane  in  the  orange  ether  extract  could 
be  purified  from  admixed  chlorophane  by  treating  the  ether  residue 
with  petroleum  ether  (not,  however,  without  some  loss  of  xantho- 
phane), and  from  admixed  rhodophane  by  treating  the  chlorophane- 
free  xanthophane  with  CSg,  in  which  the  rhodophane  was  not  soluble; 
(4)  spectroscopic  examination  of  the  purified  pigments  showed  marked 
differences,  the  chlorophane  showing  two  bands,  the  other  two  pig- 
ments only  one.  It  is  difficult  to  decide  from  these  and  other  less 
important  points  mentioned,  what  kinds  of  carotinoids  are  involved. 
Save  for  the  green  color  emphasized  by  Kiihne,  one  might  be  led  to 
believe  in  the  light  of  our  present  knowledge  that  his  chlorophane  is 


142  CAROTINOIDS  AND  RELATED  PIGMENTS 

the  carotin  which  is  present  in  traces  in  fowls.  Some  basis  for  this 
is  given  by  the  fact  that  its  CS,  solution  was  orange  colored.  On  the 
other  hand,  the  spectrum  of  this  pigment,  both  in  ether  and  CS^,  as 
shown  by  Kiihne,  resembles  xanthophyll  rather  than  carotin.  Kiihne's 
xanthophane  would  seem  to  be  the  usual  xanthophyll  met  with  in 
fowls,  in  spite  of  the  one-banded  spectrum  pictured  for  it.  The  rhodo- 
phane  is  obviously  not  a  carotinoid  in  the  sense  in  which  this  term  is 
now  applied.  Whether  it  is  a  decomposition  product,  which,  in  fact, 
Waelchi  (1881)  believed  to  be  the  case  for  all  of  Kiihne's  pigments,  or 
another  type  of  pigment,  related  perhaps  to  the  carotinoids,  cannot  be 
decided  from  the  meager  evidence  at  hand.  Perhaps  this  is  the  same 
pigment  which  Wurm  (1871)  extracted  with  chloroform  from  the 
wattles  and  "roses"  (red  warty  spots  over  the  eyes)  of  pheasants,  and 
called  tetronerythrine. 

It  might  be  mentioned  in  concluding  the  reference  to  Kiihne's  work 
that  he  was  unable  to  observe  similarly  colored  globules  in  the  retinal 
epithelium  of  man,  cow,  pig  or  snakes,  but  he  did  observe  that  the 
same  three  pigments  appear  in  the  pigeon  retina  as  in  the  fowl.  Cer- 
tain observations  respecting  the  eye  pigments  of  frogs  will  be  referred 
to  presently,  in  connection  with  the  carotinoids  in  amphibia. 

Feathers.  Nowhere  among  the  vertebrates  does  pigmentation  and 
color  attain  the  brilliancy  and  variety  that  is  seen  in  the  feathers  of 
birds.  Lovers  of  bird  life,  in  general,  as  well  as  ornithologists,  have 
long  been  interested  in  the  phenomena.  The  whole  range  of  brilliant 
as  well  as  less  conspicuous  colors  seems  to  be  due  to  pigments  of  three 
colors,  namely,  red,  yellow  and  black,  together  with  the  structural 
colors  blue  *  and  white.  Various  combinations  of  these  pigments  and 
colors  appear  to  be  entirely  responsible  for  the  effects  observed.  It  is 
true  that  Gadow  (1882)  speaks  of  structural  yellow  in  birds'  feathers, 
but  the  absence  of  yellow  pigment  in  these  cases  does  not  seem  to  be 
proved.  Besides,  the  colloidal  theory  of  optical  or  structural  color 
seems  to  preclude  yellow  among  such  colors.  Of  the  three  common 
pigments  involved,  black  is  undoubtedly  melanin.  Since  the  two  re- 
maining colors  due  to  pigment  are  those  met  with  among  carotinoids, 
interest  is  at  once  aroused  as  to  the  possibility  of  the  carotinoids  being 
involved.  Unfortunately  no  modern  investigation  has  been  made  of 
these  pigments  with  this  point  in  mind,  with  the  exception  of  the 
observations  of  Palmer  and  Kempster   (1919b,  c)    showing  that  the 

*  That  blue  color  in  feathers  is  an  optical  and  not  a  pigmented  color  seems  to  have 
been  recognized  first  by  Bogdanow  (1858). 


CAROTINOIDS  IN  THE  VERTEBRATES  143 

cream  color  in  fowls  of  the  white  feathered,  normally  yellow-shanked 
breeds  is  due  to  deposits  of  xanthophyll  in  the  feathers. 

At  the  same  time  it  should  be  stated  that  Krukenberg  (1881b, 
1882a,  b,  m)  made  an  extensive  study  of  feather  pigments  from  the 
point  of  view  of  his  lipochromes,  and  inasmuch  as  most  of  his  observa- 
tions included  spectroscopic  examinations,  as  well  as  solubility  and  the 
color  reactions  with  con.  HsSO^  and  HNO3,  it  is  possible  to  draw  some 
inferences  from  his  work  which  are  of  value  in  answering  the  question 
in  hand. 

Krukenberg  confined  his  attention  almost  entirely  to  the  brightly 
colored  birds,  including  parrots,  woodpeckers,  the  birds  of  paradise, 
the  flamingo,  cardinal,  the  tigerfinch  and  bullfinch  and  numerous 
other  individual  species.  His  studies  led  him  to  distinguish  between 
five  red  pigments  and  five  yellow  pigments.  Not  all  of  these  can  be 
regarded  as  lipochromes,  even  in  the  sense  in  which  Krukenberg  used 
the  term,  and  only  a  few  can  be  considered  specifically  as  carotinoids 
with  the  evidence  given.  There  may  be  reasonable  doubt,  also, 
whether  Krukenberg  was  justified  in  considering  each  of  the  pigments 
as  separate  entities.  It  should  be  stated,  however,  that  Krukenberg, 
himself,  was  aware  of  this. 

Of  the  red  pigments,  the  most  important  were  zoonerythrine,  pre- 
viously named  by  Bogdanow  (1858)  and  rhodophane,  previously 
named  by  Kiihne  (1878).  Of  the  others,  "araroth,"  found  in  the  red, 
orange  and  yellow  feathers  of  the  great  red  macaw,  Sittace  Macao, 
and  the  yellow  and  orange  feathers  of  Aprosmictus  melanurus,  is  prob- 
ably identical  with  zoonerythrine,  as  Krukenberg,  himself,  suggested. 
The  two  remaining  red  pigments,  zoorubin  and  pseudozoorubin,  found 
in  the  male  birds,  Paradisea  papuana  and  P.  rubra,  are  not  even  lipo- 
chromes in  the  broad  sense. 

Krukenberg  believed  that  zoonerythrine  was  a  rhodophane  com- 
pound, the  character  of  which  is  not  stated.  The  properties  are  widely 
different  from  those  described  by  Kiihne  for  rhodophane,  giving  deep 
orange  solutions  in  all  the  fat  solvents,  which  readily  extracted  the 
pigment  from  the  finely  divided  feathers,  especially  after  several  days 
digestion  with  alkaline  trypsin  or  pepsin-HCl.  The  blue  color  reac- 
tion with  con.  H2SO4  was  given,  but  the  solutions  showed  no  spectro- 
scopic absorption  bands,  only  a  continuous  absorption  beginning  in 
the  green.  Because  of  this  failure  to  show  absorption  bands  one  is 
perhaps  justified  in  concluding  that  the  pigment  is  a  carotinoid,  altered 
either  by  the  animal  body  (Krukenberg,  himself,  advanced  the  idea 


144  CAROTINOIDS  AND  RELATED  PIGMENTS 

that  it  was  derived  from  the  yellow  pigment  which  colors  the  adipose 
tissue  of  many  birds)  or  by  the  methods  which  Kmkenberg  found  it 
necessary  to  use  in  extracting  the  pigment  from  the  feathers.  It  is 
well  known  that  the  spectroscopic  properties  of  the  carotinoids  are 
among  the  first  to  be  affected  adversely. 

Zoonerythrine  was  found  to  be  the  cause  of  the  color  of  the  red 
feathers  of  the  following  birds:  C alums  auriceps,  Catinga  coerulea, 
Phoenicopterus  antiquorum  (flamingo),  Cardinalis  virginianus  (the 
cardinal  bird) ,  Pyrocephalus  rubincus,  Phlegoenus  cruenta  (the  dagger- 
stab  pigeon  of  Luzon),  Trogon  Massera,  Paroaria  cucullata,  Picus 
major,  Pyrrhula  vulgaris  (bullfinch),  tigerfinch,  Megaloprepia  mag- 
niflca,  Cymbyrhynchus  makrorhynchus,  and  possibly  Ithaginus  cruen- 
tatu^.  The  red  feathers  of  the  parrots,  Eclectus  polychlorus  and 
Cacatura  roseicapilla,  contained  the  pigment  as  did  also  the  yellow 
feathers  of  the  bird  of  paradise  Xanthomelus  aureus.  In  addition, 
Krukenberg  (1882m)  lists  14  species  of  Picides  (woodpeckers)  whose 
red  pigment  is  rhodophane. 

Among  the  yellow  feather  pigments  Krukenberg  mentions  zooful- 
vine,  coriosulfurine,  paradiseofulvine,  picofulvine  and  psittacofulvine, 
believing,  as  is  evident  from  the  names,  that  the  birds  of  paradise,  the 
woodpeckers  and  the  parrots  contained,  in  some  cases,  special  yellow 
pigments  besides  the  general  ones  listed  first.  Of  these  the  special 
parrot  pigment,  psittacofulvine,  is  evidently  not  even  a  lipochrome  in 
the  broad  sense,  from  the  description  given.  Paradiseofulvine,  found 
in  the  yellow  neck  feathers  of  the  male  Diphyllodes  magnifica,  and 
the  yellow  head,  neck  and  back  feathers  of  the  male  Paradisea 
papuana  and  P.  rubra,  was  extractable  only  after  digestion  of  the 
feathers  with  alkali  or  trypsin.  Save  for  complete  absence  of  absorp- 
tion bands  it  was  identical  with  coriosulfurine.  These  facts  suggest 
that  the  treatment  necessary  to  extract  the  pigment  altered  its  spec- 
troscopic properties,  a  supposition  confirmed  by  Krukenberg's  own 
observation  that  heating  zoofulvine  in  an  alkaline  fluid  destroyed  its 
absorption  bands. 

The  properties  of  zoofulvine  and  coriosulfurine  are  so  nearly  identi- 
cal, differing  only  by  a  slight  shift  in  absorption  bands,  that  their 
separate  entity  is  very  improbable.  Krukenberg  believed  that  the 
former  was  derived  from  the  latter.  Both  pigments  were  readily  ex- 
tracted from  the  finely  divided  feathers  by  hot  alcohol  or  fat  solvents. 
Krukenberg  stated  that  coriosulfurine  withstood  saponification  better 
than  zoofulvine  but  gave  a  less  distinct  color  reaction  with  con.  H^SO^. 


I 


CAROTINOIDS  IN  THE  VERTEBRATES  145 

Both  were  very  sensitive  to  action  of  light.  The  position  of  their 
absorption  bands,  as  pictured  by  Krukenberg,  is  practically  identical 
and  resembles  xanthophyll  very  strongly.  The  identity  of  the  zooful- 
vine  and  egg  yolk  spectra  was  noted  by  Krukenberg  himself,  and  inas- 
much as  coriosulfurine  is  stated  to  be  the  pigment  found  in  the  beaks, 
shanks,  skin  and  fatty  tissue  of  fowls  and  geese,  as  well  as  in  certain 
feathers,  there  is  no  reasonable  doubt  left  that  the  two  pigments  are 
the  same  and  are  none  other  than  the  xanthophyll  met  with  in  fowls. 

The  birds  whose  feathers  owe  their  color  to  this  xanthophyll  are 
as  follows:  The  yellow  feathers  of  Euphone  nigricollis,  the  golden 
feathers  of  Oriolus  galbula,  the  yellow  feathers  of  the  canary  Fringilla 
canaria,  the  yellow  feathers  of  the  parrot  Aprosmictus  melanurus,  the 
yellow  and  green  feathers  of  Certhiola  mexicana,  and  Chlorophanes 
atricapilla,  the  green  feathers  of  the  male  parrot  Eclectus  polychlorus, 
the  orange  feathers  of  the  great  red  macaw  Sittace  Macao,  the  yellow 
ornamental  feathers  of  the  male  Paradisea  papuana,  the  yellow  and 
orange  feathers  of  Xanthomelus  aureus  and  Selencides  alba  and  the 
feathers  of  the  woodpeckers  Chrysoptilus  punctigula,  Chloronerpes 
aurulentus,  C.  Kirkii,  Dejidropicus  cardinalis,  Campethera  nubica, 
Tiga  tridactyla,  Dryocapus  auratus,  Colaptes  auratus,  and  C. 
olivaceus. 

The  yellow  picofulvine  described  by  Krukenberg  (1882m)  in  a  num- 
ber of  species  of  woodpeckers,  differs  from  the  pigments  just  described 
in  its  yellowish-green  color  in  ether  and  CHCI3,  in  its  orange  (not  red- 
orange)  color  in  CS2,  in  its  lower  solubility  in  petroleum  ether,  and  by 
the  fact  that  its  absorption  bands  are  in  a  characteristic  position, 
shifted  so  greatly  towards  the  violet  from  the  bands  of  coriosulfurine 
that  error  of  observation  seems  excluded.  One  is  reminded  strongly 
of  the  xanthophyll  (3  of  Tswett,  and  is  tempted  to  suggest,  provision- 
ally, that  a  concentration  of  this  carotinoid  in  the  feathers  of  these 
birds  is  responsible  for  the  pigmentation, 

Carotinoids  in  Fishes 

Observations  on  the  pigments  of  fishes  have  been  confined  almost 
entirely  to  those  of  the  skin,  namely,  to  the  causes  of  surface  colors. 
The  body  tissues  have  been  examined  in  only  a  few  instances.  The 
surface  colorations  may  be  likened  in  many  respects  to  the  feather 
colorations  of  birds.  The  pigments  involved  appear  to  be  almost 
wholly  reds,  yellows  and  blacks,  combined   (in  a  physical  sense)  in 


146  CAROTINOIDS  AND  RELATED  PIGMENTS 

various  ways  and  also  with  structural  blues  and  whites.  As  in  birds 
the  various  shades  of  brown  are  melanin  combinations  with  reds  and 
yellows  and  the  greens  are  combinations  of  yellow  pigment  and  struc- 
tural blues.  The  structural  whites,  however,  do  not  appear  to  be 
colloidal  phenomena  as  in  the  case  of  birds,  but  are  due  to  shiny 
crystals  of  g:uanin.  Another  marked  difference  between  the  surface 
colorations  of  birds  and  fishes  is  the  deposition  of  the  pigments  in  the 
latter  in  chromatophors  over  which  there  is  nervous  control  such  that 
a  partial  or  complete  contraction  of  the  tissues  makes  it  possible  for 
the  animal  to  undergo  marked  changes  in  color.  This  physiological 
phenomenon  is  shared  by  a  number  of  other  lower  animals,  both  among 
the  vertebrates  and  invertebrates.  One  can  find  the  whole  subject 
considered  most  exhaustively  by  Fuchs  (1914). 

As  in  the  case  of  birds  this  monograph  can  deal  only  with  the  red 
and  yellow  pigments.  It  may  be  stated  at  the  outset  that  a  most 
promising  field  for  investigation  from  the  point  of  view  of  our  present 
knowledge  of  carotinoids  is  offered  by  these  pigments.  No  investiga- 
tion whatever  has  been  undertaken  since  the  recent  developments  in 
this  field.  However,  there  is  no  reasonable  doubt  that  the  yellow  pig- 
ments, at  least,  are  carotinoids,  either  carotin  or  xanthophylls  or 
both.  What  is  needed  especially,  besides  an  exact  determination  of 
the  carotinoid  character  of  the  yellow  pigments,  is  a  study  of  the  red 
pigments  whose  solubilities  and  color  reactions  with  the  mineral  acids 
are  those  of  the  carotinoids,  but  which  have  failed  to  show  absorption 
bands  in  the  hands  of  previous  investigators. 

De  Merejowski  (1881)  first  called  attention  to  a  rather  widespread 
occurrence  of  the  red  pigment  in  fishes,  under  the  name  of  tetronery- 
thrine.  He  later  (1883)  enumerated  some  20  species  in  which  he  had 
found  the  pigment,  in  this  paper  adopting  Bogdanow's  (1858)  name 
zoonerythrine.  No  spectroscopic  observations  were  made.  The  orange 
color  in  the  usual  fat  solvents  was  noted,  as  well  as  the  fiery  red  color 
in  CS2,  the  color  reactions  by  the  strong  mineral  acids,  and  the  bleach- 
ing in  the  air  and  sunlight.  It  is  interesting  that  de  Merejowski  ex- 
pressed the  opinion  that  the  same  pigment  caused  the  color  of  carrots, 
tomatoes  and  pimentoes.  Carotin,  according  to  him,  it  may  be  noted, 
is  a  water-soluble  pigment  from  carrots  and  tomatoes. 

Krukenberg  (1881a)  first  noticed  the  red  zoonerythrine  in  fishes  in 
the  tailfin  of  Luvarus  imperialis,  the  microscope  showing  the  red 
granular  deposits  in  the  epithelial  cells.  On  extraction  with  fat  sol- 
vents  or    hot    alcohol   the   pigment    confirmed   the    observations   of 


CAROTINOIDS  IN  THE  VERTEBRATES  147 

de  Merejowski,  and  in  addition  failed  to  show  any  absorption  bands. 
Krukenberg  (1882e)  later  found  the  same  pigment  in  the  skin  of  the 
goldfish,  Cyprinus  auratus  and  Cyprinus  Carpio  and  (1882n)  in  the 
skin  of  Mullus  barbatics.  The  red  lipochrome  in  the  latter  fish,  and 
that  which  Krukenberg  and  Wagner  (1885)  extracted  from  the  red 
salmon  muscle,  yielded  a  pigment  on  saponification  which  showed  the 
single  absorption  band  at  F  of  Kiihne's  rhodophane.  It  will  be  remem- 
bered that  Krukenberg  found  the  same  pigment  in  the  feathers  of 
certain  birds.  MacMunn  (see  Cunningham  and  MacMunn,  1883) 
later  found  it  in  a  number  of  other  fishes.  In  color,  the  pigment 
resembles  carotin  most  closely.  Its  relation  to  this  pigment  should  be 
determined. 

Krukenberg  (1882e,  n)  first  noted  the  yellow  pigments  in  fishes, 
extracting  them  from  Cyprinus  carpio,  where  they  were  present  in  the 
skin  along  with  the  red  chromolipoid,  and  from  the  skin  of  Barbus 
fluviatilis,  Muraena  Helena,  Belone  rostrata,  Scorpoena  scroja,  where 
they  existed  free  from  red  pigment,  and  from  Mullus  barbatus,  which 
contained  the  red  pigment,  as  already  noted.  The  absorption  spectra 
of  these  pigments  show  their  carotinoid  nature,  but  it  is  difficult  to 
decide  whether  carotin  or  xanthophyll  is  the  predominating  pigment, 
in  view  of  the  possibility  that  both  types  of  carotinoid  were  present  in 
the  solutions. 

The  much  more  extensive  observations  of  MacMunn  (Cunningham 
and  MacMunn,  1893)  on  the  chromolipoids  of  the  skins  of  a  number 
of  other  species  of  fishes,  which  are  accompanied  by  measurements 
of  the  absorption  bands  in  ether,  chloroform  and  carbon  disulfide,  are 
somewhat  more  instructive.  A  comparison  of  the  data  with  known 
measurements  of  the  absorption  bands  of  the  carotinoids  leads  to  the 
following  tentative  conclusions:  Carotin  is  the  chief  carotinoid  in  the 
skin  of  the  Flounder  {Pleuronectes  flesus),  the  Plaice  (P.  platessa), 
the  Dab  (P.  limanda),  the  Merry  Sole  (P.  microcephalus) ,  of  Solea 
variegata,  and  of  the  Smelt  {Osmerus  eperlanus) ;  xanthophyll  is  the 
chief  carotinoid  in  the  skin  of  Arnoglossus  megastoma,  Trigla  cuculus, 
Trigla  hirundo,  the  Mackerel  {Scomber  scombrus) ,  Syngnathus  acus, 
Siphonostoma  typhle,  Clupea  narengus,  Artherina  presbyter,  the  John 
Dorey  [Zeu^  faber)  and  the  fifteen  spined  Stickleback  (Gasterosteus 
spinachia) ;  both  carotin  and  xanthophyll  are  found  in  Cottus  bubalis, 
and  the  banded  Pipe  Fish  {Nerophis  oequoreus) ;  a  pigment  whose 
spectra  strongly  resembled  lycopin  is  the  cause  of  the  skin  pigment 
of  the  goldfish,  Cirassius  auratus. 


148  CAROTINOIDS  AND  RELATED  PIGMENTS 

One  cannot  leave  the  paper  of  Cunningham  and  MacMunn  without 
referring  to  the  interesting  experiments  of  Cunningham  on  the  color- 
less side  of  flounders.  These  interesting  fish,  as  is  well  known,  have 
the  habit  of  lying  continuously  on  their  left  sides  as  near  as  possible 
to  the  bottom  of  the  sea,  or  the  tank  in  which  they  may  be  placed. 
The  lower  side  of  the  fish  is  almost  always  devoid  of  the  black  and 
yellow  color  which  characterizes  the  upper  side  of  the  fish.  Cunning- 
ham, however,  was  able  to  cause  the  fish  to  develop  normal  pigmen- 
tation on  both  sides  by  placing  them  in  a  tank  with  a  glass  bottom 
with  a  light  reflecting  mirror  below  it  so  that  the  fish  were  exposed 
to  daylight  on  both  sides.  Cunningham  and  MacMunn  naturally  con- 
cluded that  it  is  light  which  causes  the  deposition  of  pigment  in  the 
flounder's  skin.  There  seems  to  be  nothing  to  discredit  this  conclusion 
so  long  as  one  accepts  as  proved  that  pigment  is  actually  absent  from 
the  colorless  side  of  the  flounder  and  that  chromatophors  in  the 
epithelial  tissues  play  no  part  in  the  phenomenon. 

With  regard  to  chromolipoids  in  other  tissues  of  the  fishes,  informa- 
tion is  almost  completely  lacking.  We  have  the  observations  of 
Krukenberg  and  Wagner  (1885)  already  referred  to,  of  a  red  zoonery- 
thrine  in  salmon  muscle,  changing  to  a  rhodophane  on  saponification. 
We  also  have  the  statement  of  MacMunn  (1883)  that  the  liver  of 
fishes  may  contain  a  tetronerythrine  (zoonerythrine) .  Finally,  we 
have  Miss  Newbigin's  (1898)  examination  of  the  red  pigment  in 
salmon  muscle,  m  which  she  found  a  yellow  non-lipochrome  pigment 
as  well  as  the  red  lipochrome,  showing  the  usual  lipochrome  reactions 
save  the  absorption  bands.  She  believed  that  the  red  pigment  readily 
formed  compounds  with  sodium  and  potassium,  which  could  be  decom- 
posed with  acetic  acid.  The  yellow  pigment  was  soluble  in  the  fat 
solvents',  did  not  form  compounds  with  sodium  and  potassium  but 
failed  to  show  the  color  reactions  with  concentrated  acids.  The  spec- 
troscopic absorption  properties  apparently  were  not  observed.  The 
same  red  and  yellow  pigments  were  also  present  in  the  ovaries  of  the 
mature  female. 

Carotinoids  in  Amphibians 

The  phenomena  governing  the  coloration  of  these  vertebrates,  as 
well  as  the  colors  observed,  are  almost  identical  with  those  of  fishes. 
As  in  the  case  of  the  fish  pigments,  the  chromolipoids  offer  an  inter- 
esting problem  for  study  from  the  newer  point  of  view  of  these  pig- 
ments.   The  observations  which  have  been  made  from  the  older  lipo- 


CAROTINOIDS  IN  THE  VERTEBRATES  149 

chrome  point  of  view  have  been  confined  to  the  frog  and  salamander. 

The  alcohol  and  ether  extractability  of  the  yellow  pigment  in  frogs 
was  known  to  the  early  observers,  such  as  v.  Wittich  (1854),  Leydig 
(1868),  Hering  and  Hoyer  (1869),  before  Capranica  (1877)  found 
that  the  retinal  pigment  of  the  frog  corresponded  in  its  general  solu- 
bility, chromatic  and  spectroscopic  properties  with  the  corpus  luteum 
and  egg  yolk  pigment.  Kiihne's  (1878)  chromophane  studies  included 
the  pigment  in  the  retinal  and  adipose  tissue  of  frogs,  as  well  as  the 
skin.  Only  one  pigment  was  found,  readily  and  completely  extract- 
able  from  the  saponified  extracts  with  petroleum  ether.  On  account 
of  a  slight  spectroscopic  difference  from  the  pigment  of  egg  yolk 
(absence  of  a  faint  third  band  in  spectrum  of  frog  pigment)  Kiihne 
gave  the  frog  pigment  the  name  lipochrin. 

Krukenberg  (1882c)  repeated  Kiihne's  work  on  the  yellow  or  orange 
skin  pigment  of  the  frogs  Hyla  arborea,  Rana  esculenta,  the  toads 
Bujo  viridis,  Bufo  calamita,  Bufo  vulgaris,  and  the  orange  skin  pig- 
ment of  the  salamanders,  Triton  cristatus  and  Salamdra  maculosa. 
The  same  pigment  was  found  throughout,  also  in  the  ovaries  of 
B.  calamita  and  the  fatty  tissue  of  Triton  cristatus.  A  comparison  of 
the  spectral  drawings  of  Kiihne  and  Krukenberg  for  their  amphibian 
lipochromes  shows  certain  differences  in  the  positions  of  the  absorption 
bands  such  that  it  is  impossible  to  decide  whether  the  pigment  is 
carotin  or  xanthophyll,  so  that  the  determination  of  this  important 
point  will  have  to  be  left  to  future  investigation.  It  should  be  stated, 
perhaps,  that  Magnan  (1907a,  b)  has  claimed  to  have  isolated  a  green 
and  a  yellow  pigment  from  several  Batracian's  skins,  the  yellow  pig- 
ment differing  from  the  chromolipoid  obtained  by  previous  workers 
in  that  it  failed  to  show  absorption  bands,  and  was  soluble  in  NaOH 
and  KOH.  One  cannot  help  but  raise  some  doubt  as  to  the  accuracy 
of  this  worker's  observations  both  as  to  the  properties  of  his  yellow  pig- 
ment and  the  existence  of  a  green  pigment.  It  is  an  old  observation 
that  the  frog  skin  loses  its  green  color  on  extraction  of  the  yellow 
pigment,  showing  that  the  green  color  is  partly  of  pigment  and  partly 
of  structural  origin. 

Carotinoids  in  Reptiles 

The  surface  colorations  of  reptiles  are  perhaps  even  more  conspicu- 
ous than  those  of  amphibians.  The  lizards  and  snakes  have  been 
studied  most,  and  there  has  been  at  least  one  observation  regarding 
lipochromes  in  turtles.    The  deposition  of  the  skin  pigment  in  nerve 


150  CAROTINOIDS  AND  RELATED  PIGMENTS 

controlled  chromatophores  and  the  consequent  power  to  change  color 
has  been  developed  to  a  high  point  of  perfection  in  the  lizards. 

Among  the  snakes,  which  frequently  are  marked  with  yellow  colors, 
lipochromes  in  the  broad  sense  and  thus  carotinoids,  in  the  narrower 
sense,  do  not  appear  to  occur.  Kiihne  (1878)  noticed  the  absence  of 
retinal  pigments  in  snakes.  According  to  Krukenberg  (1882d)  the 
yellow  pigment  which  can  be  extracted  after  long  boiling  with  absolute 
alcohol  from  the  skin,  muscles,  connective  tissue  and  fatty  tissue  of 
the  snakes,  Tropidonotus  natrix,  Elaphis  quadrilineatis  Bonaparte, 
Callopeltis  quadrilineatis  Pallas  and  Rhinescis  scalaris,  and  which  is 
soluble  is  ether,  CHCI3  and  CSg  after  extraction,  differs  from  the 
lipochromes  in  the  persistent  green  fluorescence  of  its  solutions,  the 
failure  to  show  absorption  bands  or  chromatic  reactions,  and  the 
failure  to  bleach  with  oxidizing  agents.  Similarly,  according  to  Cun- 
ningham and  MacMunn  (1893)  the  yellow  skin  pigment  of  the  alliga- 
tor is  not  lipochrome. 

Among  the  lizards,  however,  the  presence  of  ether  and  alcohol 
soluble  pigments  of  yellow  color  was  apparently  observed  by  a  num- 
ber of  workers  before  Krukenberg  (1882d,  n)  first  submitted  them 
to  spectroscopic  examination.  Using  the  skins  of  the  cameleons 
Lacerta  muralis,  Lacerta  agilis,  Camaelon  vulgaris  and  Bombinator 
igneus,  the  yellow  and  orange  pigments  were  extracted  and  found  to 
correspond  completely  with  other  yellow  and  orange  lipochromes. 
The  position  of  the  two  absorption  bands  resembled  most  those  previ- 
ously found  by  Krukenberg  for  the  feather  pigment  zoofulvine,  but 
because  of  an  uncertainty  in  his  mind  as  to  the  identity  of  the  pig- 
ments, Krukenberg  called  the  lizard  pigment  lacertofulvine.  In  all 
probability  the  pigment  is  xanthophyll,  or  at  least  one  of  this  group  of 
carotinoids. 

Among  turtles  we  have  the  observation  of  Halliburton  (1886)  that 
the  blood  serum  and  adipose  tissue  of  the  tortoise  is  rich  in  a  lipo- 
chrome showing  the  spectroscopic  and  other  lipochromatic  characters 
of  the  blood  serum  and  adipose  tissue  pigments  of  the  hen.  The  ques- 
tion needs  further  study,  however,  before  it  can  be  even  regarded  as 
probable  that  this  pigment  is  xanthophyll. 

Summary 

Piccolo  and  Lieben  (1866)  and  Holm  (1867)  isolated  the  first  ani- 
mal chromolipoid  in  pure  condition,  namely,  the  pigment  of  the  corpus 


CAROTINOIDS  IN  THE  VERTEBRATES  151 

luteum  of  the  cow.  Although  the  general  relation  of  this  pigment  to 
other  yellow  animal  pigments,  and  even  to  certain  of  the  plant 
chromolipoids  was  recognized  by  a  number  of  subsequent  investigators, 
its  identity  as  carotin  was  not  established  until  the  work  of  Escher 
(1913).  The  character  of  the  corpus  luteum  pigment  in  other  mam- 
mals and  in  man  has  not  been  determined.  Carotinoids  are  absent 
entirely  in  the  case  of  the  so-called  yellow  bodies  on  the  ovaries  of 
swine. 

The  existence  of  a  chromolipoid  in  the  blood  serum  of  certain 
mammals  was  known  as  early  as  1835.  Krukenberg  (1885a)  first 
succeeded  in  isolating  the  pigment  (using  ox  serum)  and  classified  the 
pigment  as  a  lipochrome.  The  relation  of  the  pigment  to  the  caro- 
tinoids which  characterize  other  mammalian  tissues  was  not  estab- 
lished until  the  work  of  Palmer  and  Eckles  (1914c).  The  chromo- 
lipoid of  cattle  and  horse  serum  is  carotin,  but  in  man  it  may  be  either 
carotin  or  xanthophyll.  Carotin,  when  present,  is  frequently,  if  not 
always,  bound  to  colloidal  serum  albumin,  but  this  does  not  appear 
to  be  the  case  for  xanthophyll.  Carotinoid  is  not  always  the  sole 
pigment  in  blood  serum,  bilirubin  also  being  present  at  times,  particu- 
larly in  the  case  of  man  and  the  horse.  The  blood  serum  of  a  num- 
ber of  mammals  is  almost  or  entirely  devoid  of  carotinoid  pigment 
under  all  conditions,  e.g.,  swine,  sheep,  goats,  dogs,  cats,  guinea  pigs 
and  rats.  This  is  also  true  for  the  new-born  animals  of  the  species 
whose  serum  is  pigmented  in  later  life. 

The  chromolipoid  of  milk  fat  is  the  carotinoid  which  characterizes 
the  blood  plasma  of  the  animal,  as  shown  by  the  author's  studies. 
Carotinoid  coloration  of  milk  fat  is  not,  however,  universal  among 
mammals,  pigmentation  being  determined  by  the  kind  and  amount  of 
carotinoid  carried  by  the  blood. 

The  chromolipoids  of  the  adipose  tissue,  internal  organs,  nerve  cells 
and  skin  of  mammals  are  the  carotinoids  which  characterize  the  blood 
serum,  only  those  animals  whose  blood  serum  is  normally  pigmented 
with  carotinoids  depositing  the  pigments  in  their  body  tissues  and 
organs. 

The  more  frequent  observation  of  an  epidermal  carotinoid  coloration 
among  diabetics  than  among  well  persons  is  due  largely  to  the  vege- 
tarian character  of  the  diet  in  diabetes,  from  which  the  pigments  are 
derived.  The  author  suggests,  however,  that  the  phenomenon  is  due 
also,  in  part  at  least,  to  the  lowered  oxidative  tone  of  the  body  in  this 


152  CAROTINOIDS  AND  RELATED  PIGMENTS 

disease,  inasmuch  as  oxidation  is  undoubtedly  the  normal  means  by 
which  the  animal  body  destroys  surplus  carotinoids. 

The  chromolipoid  pigments  of  birds  offer  many  interesting  physio- 
logical problems,  particularly  because  of  -  their  chemical  difference 
from  the  chromolipoids  of  mammals.  The  egg  yolk  pigment  was 
studied  as  early  as  1867,  and  while  Kiihne  (1878)  first  recognized  that 
it  is  not  identical  with  the  pigment  of  the  corpus  luteum  of  mammals, 
its  probably  chemical  relation  to  plant  xanthophyll  was  not  suggested 
until  the  work  of  Schunck  (1903).  This  relation  was  established  by 
Willstatter  and  Escher  (1912),  and  extended  to  include  a  biological 
relation  by  Palmer  (1915).  Whether  the  hen's  body  modifies  slightly 
the  plant  xanthophyll  or  selects  one  of  the  several  plant  xanthophylls 
differing  in  melting  point  from  the  mixed  product  obtained  from  green 
leaves,  cannot  be  decided  at  present. 

Xanthophyll  also  appears  to  be  the  chief,  if  not  the  sole  carotinoid 
in  the  blood  serum,  adipose  tissue,  nerve  cells,  body  organs  and  skin 
of  fowls.  Detailed  studies  have  not  been  made  for  other  birds.  The 
relation  of  the  so-called  lipochromes  of  the  retina  of  the  eyes  of  birds 
to  the  carotinoids  is  indefinite. 

Carotinoids  and  related  pigments  are  unquestionably  the  cause  of 
the  yellow  to  red  color  of  the  feathers  of  certain  birds  which  are 
enumerated  in  the  text.  Although  these  pigments  have  not  been 
studied  since  the  recent  advances  in  our  knowledge  regarding  animal 
carotinoids,  the  evidence  points  to  the  fact  that  xanthophyll  is  one 
of  the  pigments  concerned  in  feather  coloration. 

Skin  coloration  of  fishes  is  similar  in  some  respects  to  feather 
coloration  in  birds  except  that  the  structural  whites  are  not  colloidal 
in  fishes  and  fish  pigments  are  deposited  in  chromatophores  over  which 
there  is  physiological  control.  The  red  chromolipoid  of  the  skin  of 
many  fishes  does  not  appear  to  be  identical  with  any  of  the  known 
carotinoids  although  it  resembles  carotin  in  many  respects  and  appears 
to  be  related  to  the  red  so-called  carotinin  found  in  many  lower  plants. 
The  yellow  chromolipoids  of  fishes  are  no  doubt  true  carotinoids,  the 
evidence  available,  based  on  older  observations  of  Krukenberg  and 
MacMunn,  indicating  the  presence  of  both  carotin  and  xanthophylls. 
MacMunn  has  even  described  a  lipochrome  in  the  skin  of  the  gold- 
fish Cirassius  auratus,  which  strongly  resembles  lycopin. 

The  chromolipoids  of  the  blood,  body  tissues  and  organs  of  fishes 
have  been  examined  only  in  the  case  of  the  salmon,  in  which  the  red 


i 


CAROTINOIDS  IN  THE  VERTEBRATES  153 

pigment  of  the  flesh  and  ovaries  appears  to  be  the  carotinoid-like 
carotinin  described  above. 

Among  amphibians  the  yellow  pigments  of  the  skin,  adipose  tissue 
and  retina  of  frogs,  toads  and  salamanders  are  unquestionably  caro- 
tinoids,  but  the  evidence  at  hand  does  not  show  whether  carotin  or 
xanthophyll  or  both  are  concerned. 

Yellow  pigments  among  reptiles  appear  to  be  more  frequently  non- 
carotinoid  in  nature.  There  is  a  probability,  however,  that  a  xantho- 
phyll is  the  chromolipoid  of  cameleon  skins.  The  blood  serum  and 
adipose  tissue  of  the  tortoise  also  contain  carotinoids,  the  nature  of 
which  is  not  known. 


Chapter  V 
Garotinoids  in  Invertebrates 

The  causes  of  the  colorations  and  pigmentations  encountered  among 
the  lower  forms  of  animal  life  have  not  been  without  interest  to 
the  biologists.  It  is  to  be  expected  that  the  developments  in  the 
field  of  chromatology  which  took  place  during  the  19th  century  should 
be  accompanied  by  studies  of  invertebrate  pigments  by  the  zoologists 
and  others  interested  in  these  forms  of  life.  These  studies  have  an 
especially  important  bearing  on  the  subject  of  the  distribution  of 
carotinoid  pigments  among  animals  because,  as  has  already  been 
pointed  out,  evidence  of  a  more  definite  nature  has  been  presented  for 
the  existence  of  these  plant  pigments  in  animals  of  the  invertebrate 
group,  than  for  a  number  of  the  vertebrates.  It  should  be  under- 
stood, however,  that  carotinoids  do  not  predominate  among  the  pig- 
ments of  the  lower  animals.  On  the  contrary,  one  would  hardly  be 
justified  in  asserting  that  the  carotinoids  predominate  among  the  pig- 
ments of  yellow  to  red  color  encountered  among  the  invertebrates. 
As  in  the  case  of  plants,  it  appears  that  as  one  descends  the  scale  of 
living  forms,  non-carotinoid  pigments  of  yellow  to  red  hues  seem  to 
be  met  more  and  more  frequently.  For  example,  it  will  be  shown 
presently  that  carotinoids  are  undoubtedly  abundantly  present  in  the 
larvae  and  pupae  of  butterflies  and  moths,  but  the  brilliant  reds,  golds 
and  yellows  seen  in  the  butterflies  themselves  are  apparently  caused 
by  pigments  of  entirely  different  characteristics.  Again,  among  the 
Crustacea  and  worms,  other  red  and  yellow  pigments  are  often  the 
cause  of  colorations.  These  facts,  however,  do  not  detract  from  the 
interest  which  is  naturally  aroused  by  the  presence  of  the  carotinoids 
in  at  least  some  species  of  almost  all  the  main  groups  of  invertebrate 
animals.  One  might  think,  perhaps,  that  the  simpler  digestive  appa- 
ratus of  the  lower  animals  would  insure  a  more  abundant  distribution 
of  biologically  derived  pigments.  Whether  this  is  true  or  not  will  have  • 
to  be  decided  by  the  investigations  of  the  future. 

154 


CAROTINOIDS  IN  INVERTEBRATES  155 

Carotinoids  in  Insects 

Zoologists  recognize  as  many  as  eleven  different  orders  of  the 
Insectia,  and  state  that  a  million  species — more  or  less — may  exist  in 
the  world.  When  these  figures  are  contrasted  with  the  fact  that  not 
over  thirty-five  or  forty  species,  belonging  to  four  orders,  have  been 
examined,  with  reasonable  indications  of  carotinoids  or  closely  related 
pigments  being  present,  it  is  seen  that  very  little,  indeed,  has  been 
done  in  this  field. 

The  insect  orders  in  which  carotinoids  appear  to  be  present  are  the 
Lepidoptera  (butterflies),  Rhynchota  (bugs),  Coleoptera  (beetles) 
and  the  Orthoptera  (locusts,  grasshoppers). 

Lepidoptera.  In  the  butterflies  themselves  the  brilliant  wing  colors 
are  not  due  to  carotinoids,  as  already  mentioned.  There  are  undoubt- 
edly some  color  effects  which  are  purely  structural,  but  the  red,  orange, 
and  yellow  pigments  appear  to  be  derivatives  of  uric  acid,  as  shown 
by  the  investigations  of  Hopkins  (1889,  1891,  1892,  1896)  and  Urech 
(1893).  In  the  larvae  and  pupae,  however,  either  carotinoids  or 
modified  carotinoids  are  frequently  encountered. 

Medola  (1873)  first  showed  that  the  green  color  of  insects  is  not 
due  merely  to  the  green  digestive  mass  in  the  food  canal,  but  to  a 
true  absorption  of  pigment  by  the  hamolymph  (blood)  of  the  animals, 
although  in  a  somewhat  modified  form.  This  laid  the  foundation  for 
the  classic  experiments  of  Poulton  (1885)  on  the  pigments  of  the 
larvae  and  pupae  of  a  number  of  species  of  butterflies.  Poulton  dis- 
tinguished between  two  kinds  of  pigments  in  phytophagus  larvae, 
namely,  those  derived  from  the  food  and  those  produced  by  the  ani- 
mals themselves.  The  general  thesis  which  his  work  supports  may 
perhaps  best  be  explained  by  the  following  quotation.  "All  green 
coloration  is  due  to  chlorophyll;  while  nearly  all  yellows  are  due  to 
xanthophyll.  All  other  colors  (including  black  and  white,  and  some 
yellow,  especially  those  with  an  orange  tinge)  are  due  to  the  second 
class  of  cause  (so  far  as  I  am  aware:  It  is,  however,  extremely  prob- 
able that  certain  colors  may  be  proved  to  arise  from  the  modification 
of  the  derived  pigments,  and  many  observations  make  it  probable  that 
other  colors  may  be  derived  from  plants  in  the  case  of  larvae  feed- 
ing upon  petals,  etc.).  The  derived  pigments  often  occur  dissolved 
in  the  blood,  or  segregated  in  the  subcuticular  tissues  (probably  the 
hypodermis  cells),  or  even  in  the  chitinous  layer,  closely  associated 
with  this  cuticle  itself." 


156  CAROTINOIDS  AND  RELATED  PIGMENTS 

Our  interest  naturally  centers  around  the  derived  "xanthophylls" 
found  in  the  hamolymph  and  other  tissues,  and  Poulton's  proof  for 
its  existence.  It  may  be  stated  first,  however,  that  Poulton  used  the 
word  xanthopyll  in  a  collective  sense  for  the  yellow  pigments  accom- 
panying chlorophyll.  The  use  of  the  word  carotinoids  conveys  the 
same  meaning.  The  proof  for  the  derived  carotinoids  in  the  larvae 
and  pupae  rested  largely  upon  a  spectroscopic  examination  of  the 
blood  extracts  in  comparison  with  the  spectrum  of  the  pigments  of 
green  leaves  under  like  conditions.  Poulton's  own  conclusion  was 
that  the  points  of  difference  between  the  derived  "xanthophyll"  spec- 
trum of  caterpillars  and  that  of  green  plants  made  it  impossible  to 
decide  whether  more  than  one  derived  "xanthophyll"  was  present. 
Interpreted  from  the  point  of  view  of  our  present  knowledge  of  the 
carotinoids  this  means  that,  inasmuch  as  Poulton  was  dealing  with 
extracts  for  which  no  purifications  were  attempted,  it  is  impossible  to 
decide  whether  carotin  or  xanthophyll  or  a  mixture  of  carotinoids 
causes  the  colors  of  the  carotinoid  type  found  in  caterpillars. 

It  is  impossible  to  review  Poulton's  entire  paper.  There  is  one 
further  point,  however,  which  may  throw  some  light  on  the  character 
of  the  carotinoids  taken  up  by  these  insects  and  which  at  least  forms 
an  interesting  link  between  carotinoids  as  found  in  mammals  and  the 
same  pigments  in  caterpillars.  This  point,  is  the  great  stability  of 
the  "xanthophyll"  in  the  blood  of  these  insects,  which  led  Poulton  to 
believe  that  it  "may  be  due  to  association  with  a  protein  of  the 
blood."  Poulton  found  that  ether,  chloroform  and  carbon  disulfide 
would  not  extract  the  pigment  from  the  blood,  although  the  ether 
precipitated  the  blood  proteins  in  the  form  of  a  green  jelly  and  even- 
tually, after  some  hours,  became  bright  yellow  with  pigment.  How- 
ever, when  alcohol  was  used  as  a  protein  precipitant,  the  pigments 
dissolved  at  once  in  the  supernatant  alcohol,  especially  if  absolute 
alcohol  was  employed.  If  the  affinity  of  carotin  for  blood  protein,  as 
found  in  some  cases  for  mammals,  is  a  universal  property  of  this 
pigment,  these  results  of  Poulton's  on  the  haemolymph  of  caterpillars 
lend  support  to  the  tentative  conclusion  that  the  chief  carotinoid  of 
the  larvae  and  pupae  of  butterflies  is  carotin. 

In  view  of  the  small  number  of  insect  species  studied  from  the  point 
of  view  of  carotinoids  it  may  be  well  to  mention  that  the  species 
of  Lepidoptera  examined  by  Poulton  were  as  follows:  Pygaera 
Bucephalus,  P.  Meticulosa,  Smirinthus  Tilioe,  S.  Populi,  S.  Oscellatu^, 


CAROTINOIDS  IN  INVERTEBRATES  157 

Sphinx  Ligustru,  C.  Elpenor,  D.  Vinula,  Papilio  Machaon,  Ephyra 
Punctaria,  and  E.  Angularia. 

Krukenberg  (1886)  made  spectroscopic  observations  of  the  pig- 
ments in  the  haemolymph  of  the  pupa  of  several  additional  species  of 
Lepidoptera,  namely,  Platisama  Cocropia,  Telea  Polyphemus,  Satur- 
nia  Pemyi  and  Saturnia  Pyri.  In  these  cases  the  haemolymph  itself, 
as  well  as  the  alcoholic  extracts,  showed  the  lipochrome  absorption 
bands. 

In  the  case  of  Saturnia  Pyri  lipochrome  was  also  extracted  from  the 
body  tissues.  The  hamolymph  of  another  species,  Collosamia  Pro- 
methia,  did  not  show  the  presence  of  lipochrome  until  first  extracted 
with  alcohol.  Krukenberg's  observations,  unfortunately,  do  not  throw 
any  light  on  the  character  of  the  carotinoids  present  in  caterpillars, 
although  they  support  strongly  the  idea  of  a  general  distribution  of 
carotinoids  in  the  blood  and  tissues  of  these  herbivorous  insects.  This 
cannot  be  said,  however,  of  the  recent  extensive  study  of  this  question 
by  Geyer  (1913).  According  to  Geyer's  own  conclusions  his  results 
are  in  entire  agreement  with  Poulton's  as  to  the  presence  of  xantho- 
phyll  in  the  haemolymph  of  the  larvae  and  pupae  of  the  Lepidoptera. 
In  spite  of  an  acknowledged  familiarity  with  the  work  of  Willstatter, 
Geyer  compared  the  spectrum  of  ether  solutions  of  the  haemolymph 
pigments  with  extracts  of  yellow  flowers  obtained  with  70  per  cent 
alcohol.  Inasmuch  as  this  solvent  does  not  extract  the  true  caroti- 
noids from  plant  tissues,  it  is  not  surprising  that  Geyer's  spectrum 
revealed  no  absorption  bands  in  the  blue  and  green.  His  blood  ex- 
tracts also  failed  to  show  absorption  bands,  in  opposition  to  the  work 
of  Poulton,  so  that  we  are  still  in  the  dark  as  to  the  exact  nature  of 
the  carotinoid  pigments  taken  up  by  the  caterpillars.  This  is  unfor- 
tunate because  Geyer's  work  is  sufficiently  recent  to  have  permitted 
him  to  use  the  technic  which  would  have  given  the  desired  information. 

While  Geyer's  studies  are  disappointing  with  respect  to  the  kind  of 
carotinoids  present  in  caterpillars,  he  noticed  an  interesting  sexual 
difference  among  certain  species  in  connection  with  the  pigmentation 
of  the  hamolymph.  In  Bombyx  mori,  the  larva  and  pupa  blood  of 
the  males  was  always  colorless  or  very  faintly  tinted,  while  that  of  the 
females  was  always  a  bright  golden  yellow.  Similarly  in  the  females 
of  Xanthia  flavago  the  blood  was  yellowish  green,  while  that  of  the 
males  was  colorless  or  very  pale  yellow.  In  other  species  the  blood  of 
the  females  was  green,  containing  both  chlorophyll  and  carotinoids,  but 
the  males  again  showed  colorless  or  nearly  colorless  blood.     Geyer 


158  CAROTINOIDS  AND  RELATED  PIGMENTS 

believes  that  this  sexual  difference  is  related  to  the  ability  of  the 
females  to  impart  the  blood  pigmentation  to  the  eggs,  for  protective 
purposes,  an  idea  previously  proposed  by  Poulton,  who  also  noticed 
the  pigmentation  of  the  eggs.  Although  the  writer  is  not  in  sympathy 
with  the  protective  notions  regarding  animal  colorations,  believing  that 
such  phenomena  are  to  be  explained  entirely  on  physiological  grounds, 
and  not  through  theories  built  upon  the  assumption  that  colors  impart 
the  same  sensations  upon  the  retina  of  the  eyes  of  lower  animals  that 
they  do  upon  our  own,  it  is  nevertheless  an  interesting  fact  that  insects 
apparently  have  the  ability  to  impart  the  derived  pigments  found  in 
the  blood  to  their  eggs  just  as  is  found  in  the  case  of  the  higher  ovi- 
parous animals. 

Rynchota.  Among  this  group  of  insects,  usually  called  bugs,  red, 
yellow  and  green  colored  species  are  commonly  encountered.  Caroti- 
noids  are  to  be  expected  because  the  insects  are  mostly  phytophagous. 
Among  the  Aphids,  or  plant  lice,  the  green  colors  are  undoubtedly 
derived  from  the  food  as  in  the  case  of  caterpillar  larvse,  as  Mac- 
chiati  (1883)  first  pointed  out.  Sorby's  (1871c)  study  of  this  green 
pigment  showed,  however,  that  the  yellow  pigments  accompanying 
chlorophyll  are  also  present,  and  may  be  extracted  from  the  crushed 
insects  with  carbon  disulfide.  The  two  well-marked  absorption  bands 
in  the  blue  shown  by  these  extracts,  at  once  classifies  the  pigment 
among  the  carotinoids.  Sorby,  himself,  called  the  pigment  aphidolu- 
teine.  The  pigment  of  some  red  aphids  may  not  be  carotinoid,  be- 
cause Sorby  found  that  the  red  color  of  aphids  which  he  found  on 
apple  trees  could  be  extracted  with  hot  water,  the  extract  turning 
yellow  on  addition  of  acetic  acid,  and  red  again  when  ammonium 
hydroxide  was  added.  The  properties  of  the  pigment  suggest  an 
anthocyanin-like  substance. 

Red  coloration  in  the  tegument  among  some  species  of  bugs,  is 
unquestionably  carotinoid  at  times,  perhaps  carotin  itself.  Thus, 
Physalix  (1894)  extracted  the  red  pigment  of  the  hemipter,  Pyrrho- 
coris  a-pterus,  from  two  liters  of  the  insects.  The  pigment  was  deep 
red  in  carbon  disulfide,  yellow  in  alcohol  and  ether,  gave  the  lipo- 
chrome  reaction  with  con.  H2SO4,  and  showed  the  absorption  spectra 
of  carotin.  Physalix  asserted  that  the  pigment  was  carotin  or  a  very 
closely  related  substance, 

Coleoptera.  Green  and  yellow  and  red  pigments  also  characterize 
the  tegument  of  the  beetles.  Ley  dig  (1876)  first  noticed  the  autumn- 
like changes  in  the  color  of  the  green  beetles  Cassidce  and  the  species, 


CAROTINOIDS  IN  INVERTEBRATES  159 

Carabus  auratus,  a  phenomenon  also  noticed  by  Sorby  (1871c)  in  the 
case  of  green  Aphids.  There  can  be  little  doubt  that  the  yellow  pig- 
ments found  in  these  insects  are  'often,  if  not  always,  true  carotinoids. 
Thus,  Zopf  (1892b)  describes  the  properties  of  the  orange  pigment  in 
the  wing  coverings  of  the  willow-leaf  beetle  Clythra  quadripunctata, 
as  showing  all  the  usual  lipochrome  reactions  (including  his  lypocyan 
reaction  described  in  Chapter  III),  and  the  absorption  bands  in 
petroleum  ether  lying  at  496-480[in  and  460-448[i[x.  Zopf  called  the 
pigment  a  di-carotin  or  "eucarotin."  The  absorption  bands  indicate 
carotin  itself.  The  same  pigment  was  found  in  the  yolk  of  this  insect's 
eggs.  Schulze  (1913,  1914),  although  he  has  examined  the  pigments 
less  critically  from  a  chemical  point  of  view,  has  adopted  the  idea  that 
the  yellow  and  orange  pigments  in  the  wing  coverings  of  many  beetles 
are  true  carotinoids.  He  has  examined  especially  the  species  Mela- 
soma  XX-punctatum,  Melasoma  populi,  Chrysomela  polita  and  Chry- 
somela  varians.  He  states  (1914)  that  the  pigments  appear  to  ap- 
proach the  xanthophylls,  rather  than  carotin,  in  their  properties,  but 
does  not  present  the  chemical  evidence  for  this  statement. 

The  red  pigments  of  the  beetles  appear  to  belong  to  the  carotinoid- 
like  class  of  pigments  which  have  been  mentioned  repeatedly  in  the 
foregoing  pages  under  the  names  carotinin,  rhodophane,  zoonerythrine, 
etc.,  which  are  characterized  especially  by  showing  only  one  absorp- 
tion band  at  the  F  line.  Zopf  (1892b)  described  the  properties  of 
this  pigment  in  the  poplar  leaf  beetles  Lina  populi  and  Lina  tremulcB, 
as  well  as  the  beetles  Coccinella  septempunctata  and  C.  quinque- 
punctata.  Zopf  found  the  pigment  in  the  wing  coverings,  on  the  abdo- 
men, on  the  lateral  edges  and  end  of  the  back,  and  also  in  the  eggs 
of  the  poplar  leaf  beetles.  He  also  noticed  that  the  latter  insects 
secreted  the  pigment  from  the  mouth  when  excited  by  handling  or 
stimulated  by  chloroform.  The  Coccinellce  also  secreted  the  pigment, 
but  the  secreting  cells  were  found  to  be  in  the  joints,  not  in  the  mouth. 
Zopf  described  the  solubility  of  the  pigment  in  the  fat  solvents  and 
in  oils,  the  lipochrome  color  reactions,  including  the  lipocyan  reac- 
tion, and  the  single  spectroscopic  absorption  band  shown  by  the  ether 
solution  at  515-480[X|x.  At  the  time  of  his  investigation  of  the  pig- 
ment Zopf  called  it  a  mono-carotin,  but  later  (1893a)  referred  to  it 
as  Lina-carotin.  Griffiths  (1897)  attempted  to  ascertain  the  com- 
position of  the  pigment,  which  he  called  coleopterin,  using  the  species 
Pyrochra  coccinea,  Lina  populi  and  Coccinella  septempunctata  as 
the  source  of  his  material.    The  extracts  secured,  using  boiling  alcohol 


160  CAROTINOIDS  AND  RELATED  PIGMENTS 

or  ether,  were  purified  merely  by  repeated  re-solutions  and  evapora- 
tions. The  amorphous  residue  finally  obtained  contained  7.7  per  cent 
nitrogen,  and  showed  a  gross  composition  conforming  to  the  formula 
C7H5NOB.  This  is  the  only  analysis  that  has  been  made  of  the  sub- 
stance. The  method  employed  for  its  purification  could  not  be  ex- 
pected to  prevent  oxidation  of  the  pigment  (Zopf  showed  that  the 
pigment  readily  bleached  in  the  air)  and  would  not  insure  freedom  of 
the  product  from  alcohol  and  ether  soluble  impurities.  Griffiths'  re- 
sults are  therefore  open  to  question  and  cannot  be  accepted  as  show- 
ing the  constitution  of  this  important  carotin-like  pigment. 

Kremer  (1919)  has  recently  objected  vigorously  to  the  use  of  the 
terms  carotin  and  xanthophyll  in  connection  with  the  lipochromes  of 
the  Coleoptera,  and,  in  fact,  for  the  lipochromes  of  insects  in  general, 
on  the  grounds  that  the  older  terminology  of  Krukenberg  suffices 
until  the  character  of  the  animal  lipochromes  has  been  more  accu- 
rately determined.  It  is  agreed,  of  course,  that  all  scientific  effort 
must  advance  along  conservative  lines.  At  the  same  time  one  cannot 
afford  to  be  conservative  to  the  point  of  being  reactionary. 

Orthoptera.  The  facts  concerning  carotinoids  among  insect  pig- 
ments presented  in  the  preceding  paragraphs  in  themselves  lend  strong 
support  to  the  supposition  that  similar  pigments  exist  in  the  yellow, 
green  and  multicolored  grasshoppers  and  other  species  belonging  to 
this  group.  The  few  experimental  observations  which  have  been  made 
are,  however,  inadequate  for  the  proof  of  this  supposition.  Kruken- 
berg (1880)  recorded  a  brief  study  of  these  pigments  in  connection 
with  his  attempt  to  explain  Ley  dig's  (1876)  observation  that  the 
common  green  locust,  Locusta  viridissima,  changes  to  a  brownish- 
yellow  color  simultaneously  with  similar  changes  in  foliage  in  the 
autumn.  Although  he  found  that  the  chitinous  layers  in  this  species, 
as  well  as  Mirbius  viridis  and  the  common  green  grasshopper  con- 
tained specific  green,  yellow  and  red  pigments,  whose  varying  sensi- 
tiveness to  destruction  by  light  was  the  probable  cause  of  the  color 
changes  noted  by  Leydig,  the  chemical  properties  of  the  pigments, 
particularly  their  failure  to  show  absorption  bands,  necessarily  leaves 
the  question  open  as  to  the  probability  of  carotinoid  pigments  being 
involved  in  their  coloration.  It  is  true  that  Podiapolsky  (1907)  found 
that  a  golden  yellow  solution  was  obtained  by  treating  alcoholic  ex- 
tracts of  green  locusts  with  Ba(0H)2,  and  concluded  that  the  pigment 
thus  secured  was  apparently  identical  with  plant  xanthophyll.    Obvi- 


CAROTINOIDS  IN  INVERTEBRATES  161 

ously,  the  whole  matter  of  the  grasshopper  and  locust  pigments  needs 
further  study. 

Acerata.  The  animals  in  this  group  are  not,  strictly  speaking, 
insects,  but  are  a  lower  order  midway  between  insects  and  Crustacea. 
Heim  (1892)  examined  the  red  pigment  in  the  larvae  of  one  species, 
namely  Trombidium,  or  common  red  mite.  He  found  it  to  be  soluble 
in  the  fat  solvents  with  a  red  color  and  that  it  gave  the  lipochrome 
reaction  with  the  concentrated  acids.  Its  possible  relation  to  the 
carotinoids  is  thus  indicated. 

Carotinoids  in  Crustacea 

Pigmentation  among  the  Crustacea  is  characterized  both  by  the 
variety  of  colors  exhibited  and  by  their  brilliancy.  The  various  colors 
found,  including  blue,  green,  and  various  shades  of  orange,  red  and 
brown,  are  more  frequently  found  singly  on  a  species,  rather  than 
mixed  to  give  varied-colored  effects.  Examples  of  brilliant  single 
colors  are  seen  in  the  higher  and  lower  crabs,  the  lobster,  and  the  cray- 
fish. Instances  of  varied-colored  forms  are  the  prawns,  such  as 
Hippolyte  varians,  Leander  serrator,  and  the  wrasse,  Grenilabrus 
melops.  These  latter  species  have  various  pigments  deposited  in 
chromatophores,  whose  expansion  and  contraction  under  the  influence 
of  various  agents,  brings  about  some  remarkable  color  changes  in  the 
animals.  Contrary  to  the  situation  found  in  many  of  the  higher 
animals  the  blue  and  green  colors  encountered  in  Crustacea  are  not 
structural,  but  are  due  to  pigments  whose  relation  to  the  red  lipo- 
chrome so  common  to  these  animals  is  so  intimate  and  yet  so  fugi- 
tive, that  its  exact  nature  has  never  been  discovered. 

From  an  historical  point  of  view  Pouchet  (1876)  seems  to  have  first 
described  the  properties  of  red  and  yellow  ether  soluble  pigments  in 
the  hypodermis,  eggs  and  ovaries  of  the  lobster  and  other  Crustacea. 
Both  pigments  dissolved  in  concentrated  HgSO^  with  a  color  change 
from  green  to  blue  to  violet.  The  pigments  differed,  however,  in  that 
the  yellow  one  was  soluble  in  alcohol,  but  the  red  one  was  not.  The 
red  pigment  was  obtained  in  crystalline  form,  the  crystals  being  violet 
colored  with  a  metallic  reflection.  Jolyet  and  Regnard  (1877)  noted 
the  presence  of  a  yellow,  ether  soluble  pigment  in  crab's  blood,  and 
Frederique  (1885)  a  similar  red  pigment  in  the  blood  plasma  of  the 
lobster.  Moseley  (1877)  was  the  first  to  name  the  red  pigment,  call- 
ing it  crustaceorubin.    He  also  noticed  its  single  absorption  band  in 


162  CAROTINOIDS  AND  RELATED  PIGMENTS 

the  blue-green  part  of  the  spectrum.  Merejowsky  (1881,  1883)  de- 
scribed the  same  pigment  under  the  name  zoonerythrine,  and  enu- 
merated various  species  of  Crustacea  in  which  it  occurred.  Maly 
(1881),  working  with  the  red  eggs  of  the  spider  crab,  Maia  Squinado, 
differentiated  between  a  red  vitellorubin  and  a  yellow  vitellolutein, 
showing  many  solubility  and  chromatic  properties  in  common,  but 
differing  in  their  spectroscopic  properties  and  in  their  affinity  for 
alkalis.  Krukenberg  (1882k)  included  both  pigments  under  his  lipo- 
chromes,  but  was  convinced  of  the  identity  of  the  red  pigment  with 
Kiihne's  rhodophane.  Halliburton  (1885)  made  a  special  study  of 
the  red  "tetronerythrine"  in  the  blood  of  the  lobster,  crab  and  cray- 
fish, but  noticed  a  difference  between  the  fresh  water  {Astacus  fluvi- 
atilis)  and  salt  water  {Nephrops  norwegicus)  form  of  the  latter,  in 
that  the  pigment  was  almost  absent  from  the  salt  water  animals. 
Yellow  chromolipoid  is  not  mentioned.  MacMunn  (1883)  examined 
the  liver  and  bile  of  lobsters,  crabs  and  crayfish  for  lipochromes,  find- 
ing yellow  ''lutein"  in  some  cases  and  red  "tetronerythrine"  in  others. 
Many  of  the  investigators  mentioned  also  made  a  cursory  examination 
of  the  pigments  in  the  hypoderm,  but  these  have  been  studied  espe- 
cially by  MacMunn  (1890)  and  Newbigin  (1897)  for  the  larger 
species  of'Crustacea  and  by  Blanchard  (1890)  and  Zopf  (1893a)  for 
the  smaller.  The  latter  investigators  used  chiefly  the  little  red  Diap- 
tomus  hacillijer,  found  in  fresh  water,  for  the  source  of  their  material. 
Blanchard  found  only  one  pigment,  but  Zopf  describes  both  red  and 
yellow  pigments,  the  red  one  being  called  diaptomin. 

This  brief  historical  survey  makes  it  clear  that  two  distinct  types 
of  chromolipoids  are  present  in  Crustacea,  one  characterized  by  its  red 
color  and  the  other  bj^  a  more  yellow  hue.  How  are  these  pigments 
related  to  the  carotinoids? 

With  regard  to  the  red  pigment  its  properties  have  been  described 
most  fully  by  Maly  (1881),  Zopf  (1893a)  and  Newbigin  (1897)  as 
follows:  The  ether  and  petroleum  ether  solutions  are  yellow,  when 
dilute,  but  the  alcohol,  benzene,  chloroform  and  carbon  disulfide  solu- 
tions are  always  red  or  pink,  even  on  great  dilution.  Water,  also, 
acting  on  material  such  as  the  dried  Maia  eggs  forms  a  protein  solu- 
tion in  which  the  coloring  matter  is  apparently  dissolved,  and  from 
which  the  pigment  can  be  removed  by  coagulating  the  protein  with 
heat  or  alcohol  and  extracting  the  dried  precipitate.  The  pigment  is 
very  unstable  when  pure  and  fades  very  rapidly  in  contact  with  air, 
even  in  darkness.     This  bleaching  is  undoubtedly  an  oxidation,  and 


CAROTINOIDS  IN  INVERTEBRATES  163 

at  once  shows  the  close  relation  of  the  pigment  to  the  carotinoids. 
The  chromatic  colorations  with  the  strong  mineral  acids  are  also 
identical  with  the  carotinoid  colorations  under  like  conditions.  How- 
ever, unlike  any  of  the  known  carotinoids  it  appears  to  form  com- 
pounds with  the  caustic  alkalis,  and  alkali  earth  metals,  and  can, 
moreover,  be  precipitated  from  its  alcoholic  solution  on  addition  of 
saturated  Ba-(0H)2,  Ca(0H)2  and  Mg(0H)2  solutions.  The  pig- 
ment can  be  readily  liberated  from  its  alkali  and  alkali  earth 
compounds  by  acids,  apparently  without  injury  to  its  properties. 
According  to  Maly  and  Newbigin  these  pigment  compounds  are  insol- 
uble in  alcohol,  but  are  soluble  in  ether,  chloroform,  carbon  disulfide, 
benzene  and  petroleum  ether  (slightly).  Zopf,  however,  denied  the 
solubility  of  the  calcium  and  barium  compounds  in  any  of  these  sol- 
vents, and  states  that  the  sodium  compound,  only,  is  soluble  in  the 
solvents  mentioned.  He  noted,  also,  that  the  sodium  compound,  like 
the  free  pigment,  readily  bleaches.  Spectroscopically,  as  already  men- 
tioned, the  pigment  differs  from  the  known  carotinoids  in  that  it  shows 
only  one  absorption  band.  According  to  Zopf  this  band  in  ether  lies 
at  515-465[X[x  and  in  carbon  disulfide  at  533-482[X|j,. 

In  view  of  the  unanimity  of  the  above  mentioned  investigators  on 
the  properties  described  one  cannot  help  being  somewhat  surprised  at 
the  recent  announcement  of  Verne  (1920a,  b)  that  the  red  pigment  of 
Crustacea  is  none  other  than  carotin.  It  is  stated  that  the  pigment 
which  he  isolated  in  pure  crystalline  form  from  the  hypodermis  of 
the  Decapod  Crustacea  (lobsters,  crabs,  etc.)  has  the  same  melting 
point,  forms  the  same  iodide,  exhibits  the  same  absorption  spectra 
and  shows  the  same  composition  on  analysis  as  carotin.  It  is  true 
that  Blanchard  (1890)  called  the  impure  red  pigment  which  he  iso- 
lated carotin,  but  he  was  hardly  justified  in  so  doing  inasmuch  as  his 
pigment  showed  no  absorption  bands  whatever.  The  findings  of 
Verne,  therefore,  while  very  significant,  cannot  be  given  unqualified 
acceptance  until  it  is  possible  to  explain  the  peculiar  properties  which 
were  obtained  by  all  previous  investigators  for  this  carotin-like 
pigment. 

As  for  the  yellow  pigment  we  have  the  observations  of  Maly,  Kru- 
kenberg,  MacMunn  and  Zopf,  to  determine  its  possible  relation  to 
the  carotinoids.  Miss  Newbigin's  (1897)  failure  to  confirm  the  lipo- 
chrome  characteristics  of  this  pigment  can  only  be  explained  on  the 
grounds  that  she  was  dealing  with  a  decomposition  product.  There 
is  certainly  no  basis  for  her  idea  that  the  two-banded  spectrum  ob- 


164  CAROTINOIDS  AND  RELATED  PIGMENTS 

served  by  the  other  investigators  was  due  to  the  action  of  light  on 
the  red  pigment.  The  properties  of  the  yellow  pigment  which  show 
its  probable  carotinoid  nature  are  its  solubility  in  alcohol,  ether, 
petroleum  ether,  chloroform  and  carbon  disulfide,  its  color  being 
orange  red  in  the  last  named  solvent;  its  resistance  to  saponification; 
its  lipochrome  color  reactions  with  the  concentrated  mineral  acids; 
its  two-banded  absorption  spectrum;  and  the  great  ease  with  which 
the  pigment  bleaches.  As  to  whether  the  pigment  is  carotin  or 
xanthophyll  we  have  only  the  measurements  of  the  absorption  bands 
of  the  "lutein"  which  MacMunn  (1883)  extracted  from  the  liver  of 
the  crab.  Cancer  pagurus,  and  of  the  "yellow  carotin"  which  Zopf 
secured  from  the  little  Diaptomus  crustacean.  MacMunn  gives  the 
measurements  in  ether  as  498-480^^4,  and  466-450[xtx,  and  in  CSg  as 
530-507n^  and  496-476[X[i,.  Zopf 's  "yellow  carotin"  in  petroleum  ether 
showed  bands  at  498-479[1[a.  and  464-450[i[i.  The  agreement  exhibited 
in  like  solvents  indicates  that  these  investigators  were  dealing  with 
the  same  pigment.  The  position  of  the  bands  suggests  carotin  rather 
than  xanthophyll. 

As  is  well  known,  the  red  color  so  frequently  associated  with  Crus- 
tacea is  apparently  absent  from  the  external  tissues  until  the  appli- 
cation of  heat  produces  the  usual  brilliant  red  hue.  The  common 
lobster  is  a  conspicuous  example.  The  shell  of  this  animal  is  very 
dark  blue,  although  the  underlying  hypodermis  is  red.  In  the  case 
of  the  fresh  water  crayfish,  Astacus  nobilis,  the  shell  is  grayish  brown 
and  the  hypodermis  blue.  The  salt  water  crayfish  or  Norway  lobster, 
Nephrops  norwegicus,  has  an  orange  shell  and  red  hypodermis.  Green 
colors  are  also  seen,  as  in  the  species  Virbius  viridis.  Blue  colors 
are  found  among  the  Copepods,  also,  Merejowsky  (1883)  mentioning 
the  species  Anemalocera  Patersoni  and  Pontellina  gigantea.  The 
very  fugitive  character  of  these  blue  colors  has  been  known  for  many 
years.  Not  only  heat,  but  reagents  like  alcohol,  ether,  or  acids  change 
the  color  of  the  tissues  to  the  characteristic  red.  Pouchet  (1876) 
believed  that  the  phenomenon  was  due  to  the  destruction  of  a  very 
unstable  blue  pigment  which  then  allowed  previously  invisible  red 
pigment  to  be  seen.  Krukenberg  (1882k),  however,  advanced  the 
theory  that  the  blue  and  green  colors  were  due  to  lipochromogens 
which  were  transformed  by  the  various  reagents  into  lipochromes. 
This  theory  has  been  adopted  by  nearly  all  subsequent  investigators, 
including  Merejowsky  (1883),  MacMunn  (1890)  and  Newbigin 
(1897).    Merejowsky  called  the  lipochromogen  velelline,  after  Negri, 


CAROTINOIDS  IN  INVERTEBRATES  165 

and  describes  the  transformation  of  the  blue  aqueous  solution  into 
"zoonerythrine."  He  says,  in  substance,  that  if  a  filtered  blue  aqueous 
extract  is  treated  with  a  drop  of  acid  (HgSO^,  HNO3,  HCl,  acetic  or 
picric) ,  and  then  with  a  drop  of  strong  KOH,  NaOH  or  NH^OH,  and 
then  with  several  drops  of  absolute  alcohol,  there  is  an  instantaneous 
color  change  of  blue  to  red  orange.  On  filtering,  the  filtrate  is  color- 
less and  the  red-orange  substance  left  on  the  filter  gives  all  the  prop- 
erties of  "zoonerythrine."  Merejowsky  describes  a  similar  change 
for  a  green  water-soluble  " astro viridine"  which  he  extracted  from 
the  Crustacea,  Gebbia  littoralis  and  Paloemon  viridis. 

Miss  Newbigin  (1897)  likewise  obtained  an  aqueous  solution  of  blue 
pigment  from  the  hypodermis  of  the  lobster  and  the  epidermis  of  the 
fresh  water  crayfish  by  suspending  scrapings  from  the  shell  of  hypo- 
dermis in  0.1  per  cent  HCl.  She  states,  "This  solution  is  first  pink 
but  later  turns  blue  on  standing  as  the  solution  becomes  neutral  or 
alkaline  with  the  formation  of  CaClg  from  the  line  of  the  shell.  The 
blue  solution  is  very  unstable.  Heat  (45° — 50°  C),  acids,  alcohol  or 
ether  turn  it  pink  instantly.  The  pink  pignient  is  readily  soluble  in 
alcohol  or  ether,  and  gives  all  the  characters  of  crustaceorubin."  An 
observation  that  ammonia  is  always  given  off  at  the  conversion  of 
blue  into  red  suggested  to  Miss  Newbigin  that  the  compound  of 
lipochrome  giving  the  blue  color  is  an  organic  base.  She  points  out 
that  it  cannot  be  a  simple  ammonia  compound  because  the  alkali 
compounds  of  the  red  pigment  are  red,  not  blue. 

It  is  clear  that  the  true  explanation  of  the  character  of  these  inter- 
esting chromogens  has  not  yet  been  discovered.  One  cannot  help  but 
wonder  whether  there  may  be  an  analogy  between  these  phenomena 
and  blue  colloidal  gold.  The  sensitiveness  of  the  blue  solution  to 
reagents  which  are  known  to  aggregate  colloidal  particles  and  the 
precipitation  of  chromolipoid  which  occurs  following  the  use  of  these 
reagents  is  certainly  strongly  suggestive  of  a  colloidal  phenomenon. 
The  stabilizer  of  the  suspensoid  may  well  be  a  basic  anmionia-con- 
taining  substance  extracted  from  the  tissues  with  water. 

Carotinoids  in  Echinoderms 

The  most  familiar  of  the  animals  included  under  this  group  are  the 
Asteroids,  or  star-fishes;  the  Ophiuroids,  or  brittle  stars;  the  Echi- 
noids,  or  sea  urchins;  the  Crinoids,  or  sea  lilies;  and  the  Holothuroids, 
or  sea  cucumbers.     The  various  colors  shown  by  these  interesting 


166  CAROTINOIDS  AND  RELATED  PIGMENTS 

animals  have  been  described  by  many  zoologists.  In  general  the 
colors  of  the  echinoderms  resemble  very  closely  those  of  the  Crustacea. 
From  the  standpoint  of  the  pigments  proper  the  same  red  and  yellow 
carotinoid-like  pigments  found  in  Crustacea  appear  to  be  the  cause 
of  like  colorations,  and,  in  addition,  blue  and  green  lipochromogens 
are  also  encountered.  From  observations  cited  in  Miss  Newbigin's 
Monograph  (1898)  the  blue  and  green  colors  are  more  common  in 
species  found  in  shallow  water  than  in  the  deep-sea  forms,  where  red 
colors  predominate. 

Merejowsky  (1881)  first  called  attention  to  the  properties,  later 
ascribed  to  lipochromes,  of  the  red  pigment  in  echinoderms,  and  cited 
twenty  or  more  species,  representing  the  various  orders,  in  which  it 
occurred.  He  used  the  name  zoonerythrine  for  the  pigment,  and  later 
(1883)  reaffirmed  his  previous  observations,  especially  the  presence 
of  the  pigment  in  the  sea  cucumber  Holothuria  tubulosa,  which  had 
been  denied  by  Krukenberg.  The  observations  on  the  chromolipoids 
in  the  Holothuroids  have,  in  fact,  been  contradictory.  Krukenberg 
(1882J)  called  the  skin  pigment  of  Holothuria  Poll  uranidine,  but  later 
(1882k)  stated  that  "rhodophane"  is  present  in  an  especially  pure 
condition  in  the  ovaries  and  blood  vessels  of  this  species,  while  Mac- 
Munn  (1890)  found  no  lipochromes  in  this  species,  but  reported  a 
''rhodophane-like  lipochrome"  in  the  blood  and  "liver"  of  other  species, 
namely  Holothuria  nigra  and  H.  Ocnius  brunneus.  The  single  absorp- 
tion band  of  this  pigment  in  ether  was  placed  by  MacMunn  at  507- 
4:71n\i. 

It  is  evident  from  the  observations  of  Krukenberg  (1882k)  and 
MacMunn  (1890)  that  in  the  star-fishes,  at  least,  chromolipoids  show- 
ing two-banded  spectra  and  more  nearly  resembling  true  carotinoids 
predominate  over  the  red  pigment  showing  only  one  band.  Kruken- 
berg described  such  a  pigment  in  the  skin  and  "liver"  of  the  species 
Astrospecten  aurantiacus  and  Asteracanthion  glacialis,  under  the  name 
orangin  (on  account  of  its  color),  and  MacMunn  described  similar 
carotinoid-like  pigments  in  the  orange-colored  ovaries  of  Asterina 
gibbosa.  These  pigments  may  be  xanthophyll,  judging  from  the  ab- 
sorption spectra  described  by  Krukenberg.  It  is  evident,  however, 
that  carotin  may  be  the  cause  of  the  red,  two-banded  piginent  found 
by  MacMunn  in  the  integument  of  Goniaster  equestris,  Cribella  ocu- 
lata  and  Solaster  papposa.  The  red  ovaries  of  the  Cribella  species 
contain  the  same  pigment. 

Of  the  other  forms  of  echinoderms,  no  special  examinations  of  pig- 


CAROTIN OWS  IN  INVERTEBRATES  167 

ments  seem  to  have  been  made  for  the  brittle-stars  or  sea-urchins 
with  the  exception  of  Merejowsky's  zoonerythrine-containing  species. 
MacMunn  (1890)  reported  a  yellow  lipochrome  in  the  Crinoid,  Ante- 
don  rosacea,  but  attempts  to  ascertain  the  character  of  old  extracts 
from  other  species  of  sea  lilies  were  unsatisfactory,  as  might  be 
expected. 

The  echinoderms,  like  the  Crustacea,  may  also  contain  various  lipo- 
chromogens.  Merejowsky  (1883)  described  a  red  echinastrine,  a  green 
astroviridine,  a  gray  astrogriseine  and  a  violet  astroviolettine,  each 
soluble  in  water  and  readily  going  over  into  "zoonerythrine"  like  his 
velelline,  described  in  a  preceding  paragraph.  He  also  described  a 
brown  ophiurine  in  species  of  brittle-stars.  Ultra-microscopic  and 
ultra-filtration  studies  on  solutions  of  these  and  the  crustacean  "lipo- 
chromogens"  would  throw  some  light  on  whether  colloidal  phenomena 
are  involved,  as  was  suggested  above. 

Carotinoids  in  Molluscs 

One  does  not  ordinarily  associate  carotinoid-like  colors  with  these 
animals  among  which  are  represented  the  various  species  of  oysters, 
mussels,  snails  and  octopus.  Merejowsky  (1881,.  1883),  however,  has 
designated  a  number  of  species  of  gastropods  (snails)  and  conchiae 
among  the  ''zoonerythrine"  containing  animals.  It  is  not  stated,  how- 
ever, whether  the  pigment  is  in  the  shells,  or  in  the  animals  them- 
selves. More  specific  is  the  statement  of  Krukenberg  (1882f)  that  the 
liver  of  the  gastropod  Helix  pomatia  sometimes  contains  a  yellow 
lipochrome  showing  two  absorption  bands,  one  over  F  and  the  other 
between  F  and  G.  MacMunn  (1883,  1885a)  failed  to  find  such  a  pig- 
ment in  the  liver  of  this  species  as  well  as  a  number  of  other  gas- 
tropods, finding  only  "enterochlorophyll,"  a  pigment  showing  the 
absorption  bands  of  chlorophyll  in  the  red  and  green  parts  of  the 
spectrum,  which  MacMunn  held  to  be  of  animal  origin.  A  different 
result  was  obtained  in  the  examination  of  the  liver  of  the  mussel 
Mytilus  edulis,  in  which  a  "lutein"  pigment,  showing  an  absorption 
band  at  F  and  one  between  F  and  G  was  found  in  addition  to  the 
"enteroehlorophyll."  In  a  more  recent  study  of  the  pigments  of  mol- 
lusc livers  by.  Dastre  (1899)  there  is  described  besides  the  "chloro- 
phylloid"  (compare  with  MacMunn's  enterochlorophyll)  a  pigment 
called  cholechrome,  which  is  stated  to  be  intermediate  between  bili- 
rubin and  lipochrome.     Cholechrome,  uncontaminated  with   chloro- 


168  CAROTINOIDS  AND  RELATED  PIGMENTS 

phylloid,  is  stated  to  be  the  liver  pigment  of  Crustacea  and  other 
arthropods  (spiders,  insects). 

It  seems  to  be  apparent  from  even  these  meager  studies  that  the 
digestive  organ,  at  least,  of  molluscs  may  contain  a  pigment  which 
may  be  either  carotin  or  xanthophyll  or  a  modification  of  one  of  the 
carotinoids. 

Carotinoid  in  Worms 

Miss  Newbigin  (1898)  has  given  an  excellent  summary  of  the 
brilliant  colors,  both  pigmental  and  structural,  shown  by  the  various 
species  of  worms.  It  is  evident  that  many  types  of  pigments  are  pres- 
ent. Carotinoid-like  pigments,  however,  are  not  entirely  absent,  if 
one  is  to  judge  from  the  observations  of  the  older  investigators. 
These  observations,  unfortunately,  have  been  confined  to  only  a  few 
species  so  that  it  is  not  possible  to  decide  how  widely  distributed  these 
chromolipoids  may  be  among  the  worms. 

Krukenberg  (1882h)  found  a  rhodophane-like  lipochrome  in  the  pure 
uncontaminated  digestive  juice  of  Siphonostoma  diplochaitos.  He 
(18821)  has  also  described  a  lipochrome  in  the  cuticular  skeleton  of 
the  Polyzoa,  Bugula  neritina,  whose  spectrum  is  identical  with  that 
of  carotin.  The  pigment  is  not,  however,  the  chief  one  of  this  species. 
According  to  MacMunn  (1890)  the  orange-red  color  of  two  other 
species  of  this  class  of  worms,  namely,  Lepralia  foliacea  and  Flustra 
foUacea,  is  due  to  a  rhodophane-like  lipochrome. 

Among  the  Chaetopods,  or  segmented  bristle  worms,  MacMunn 
(1890)  found  several  species  among  the  Polychaetes  which  appar- 
ently contain  carotinoids.  In  Arenicola  piscatorium,  a  black  worm, 
the  intestine  was  found  to  be  covered  with  an  orange-colored  glandu- 
lar tissue.  The  pigment  could  be  extracted  with  the  fat  solvents  and 
alcohol,  and  the  extracts  showed  two,  possibly  three  bands  in  the 
green  and  blue.  The  integument  was  found  to  contain  the  same  pig- 
ment masked  by  melanin.  In  a  Terebella  species  the  tentacles  and 
integument  contained  a  lipochrome  showing  two  absorption  bands.  A 
like  pigment  was  found  in  the  integument  of  the  species  Cirratulus 
tentaculatus  and  C.  cirratus.  Nereis  virens,  the  common  clam  worm 
of  the  northern  seas,  contained  it  also,  but  in  smaller  quantity.  In 
Polynoe  spinifera  most  parts  of  the  worm  contained  the  same  lipo- 
chrome. 

These  observations,  while  brief,  point  very  strongly  to  the  presence 
of  carotinoids  in  worms.     Before  passing  to  the  sponges  in  which 


i 


CAROTINOIDS  IN  INVERTEBRATES  169 

carotinoid-like  pigments  appear  to  be  widely  distributed  it  might  be 
mentioned  that  there  is  no  definite  evidence  that  the  Coelenterates 
(sea-anemones,  corals,  jelly-fish  and  related  animals)  contain  caro- 
tinoids,  notwithstanding  the  brilliant  colorations  which  they  exhibit. 
It  is  true  that  Merejowsky  (1881)  listed  numerous  species  containing 
tetronerythrine,  but  Krukenberg  (1882g)  disproved  this  for  Gorgonia 
verrucosa.  MacMunn  (1890),  however,  mentioned  a  lipochrome  re- 
sembling rhodophane  or  xanthophane  in  the  red  polyp  head  of  the 
species  Tubularia  indivisa.  Further  study  is  needed  of  the  pigments 
in  this  group  of  animals. 

Carotinoids  in  Sponges 

The  Porifera,  or  sponges,  when  fresh  show  a  variety  of  colors  from 
red  to  green,  some  of  which  are  quite  brilliant,  but  others  dull.  The 
yellow,  orange  and  red  colors  have  been  found  to  be  due  almost  exclu- 
sively to  lipochromes,  in  the  broad  sense.  There  is  little  doubt  that 
the  yellow  and  orange  pigments  are  true  carotinoids.  The  red  pig- 
ment, however,  appears  to  belong  to  the  carotin-like  group  of  the 
same  color  which  is  so  widely  distributed  among  the  lower  forms  of 
animal  life. 

Krukenberg  (1880)  first  discovered  the  lipochrome  nature  of  the 
yellow  and  red  pigments  of  sponges.  It  was  not  until  he  repeated 
(18821)  his  first  observations,  however,  and  purified  his  extracts  by 
saponification  that  a  satisfactory  separation  of  the  various  pigments 
was  obtained.  The  technic  employed  was  essentially  that  used  by 
Kiihne  in  his  chromophane  studies.  The  sponges  were  extracted  with 
alcohol,  the  extract  saponified  and  the  soap  salted  out  with  NaCl. 
This  material  was  then  shaken  with  petroleum  ether  until  no  more 
pigment  was  extracted.  A  similar  treatment  with  ether  followed,  and 
if  any  pigment  remained  the  soap  was  treated  with  acetic  acid  and 
the  liberated  pigment  taken  up  with  CSg.  It  is  readily  seen  that  this 
method  would  not  lead  to  a  separation  of  carotin  and  xanthophyll. 
It  did  serve,  however,  to  separate  distinctly  carotinoid-like  pigments 
in  most  cases  from  the  rhodophane-like  chromolipoids  showing  only  a 
single  absorption  band.  In  general  the  petroleum  ether  extracts 
showed  two  absorption  bands,  and  the  residues  left  on  evaporation 
gave  the  characteristic  color  reactions  with  concentrated  acids.  The 
bands  pictured  by  Krukenberg  in  some  cases  indicate  carotin,  such 


170  CAROTINOIDS  AND  RELATED  PIGMENTS 

as  in  the  sponges  Hircinia  spinosula,  Suberites  flavus,  Tedania  Mug- 
giana  and  Suberites  massa,  while  in  others,  namely,  Papillina  suberea 
and  Tethya  Lyncureum,  xanthophyll  is  indicated.  The  ether  extract 
of  the  soap,  after  the  petroleum  ether  treatment,  in  most  cases  showed 
only  a  single  absorption  band,  from  which  Krukenberg  drew  the 
conclusion  as  to  the  presence  of  a  rhodophane-like  pigment.  Pig- 
ment of  this  character  is  apparently  not  present  in  all  sponges.  For 
example,  Krukenberg  found  only  carotinoid-like  pigments  in  Suberites 
flavus  and  Pipillina  suberea.  Other  sponges  which  contain  pigment 
showing  two-banded  spectra,  according  to  Krukenberg,  are  Reniera 
aquaeductus,  Cocospongia,  Chondrosia  reniformis,  Aplysiiia  aero- 
phoba,  and  Suberites  domuncula. 

MacMunn  (1888)  reported  spectroscopic  studies  of  the  lipochromes 
of  a  number  of  additional  species  of  Porifera,  which  throw  still  further 
light  on  the  widespread  occurrence  of  carotinoid-like  pigments  in  the 
sponges.  His  method  was  to  examine  the  alcoholic  extracts  of  the 
sponges  for  absorption  bands  and  then  shake  the  alcoholic  solution 
with  CS2  and  repeat  his  observations  on  the  CS2  solutions.  Neither 
solution  would  be  expected  to  show  the  character  of  the  carotinoids 
present  inasmuch  as  alcohol  extracts  both  classes  of  pigments  from 
tissue,  and  also  since  xanthophylls  are  partly  epiphasic  between 
alcohol  and  carbon  disulfide.  However,  if  chromolipoid  pigments 
remained  in  the  alcohol  after  the  carbon  disulfide  extraction,  this  fact 
would  indicate  the  presence  of  xanthophylls. 

Almost  all  of  MacMunn's  observations  of  absorption  bands  of  the 
lipochromes  in  both  alcohol  and  carbon  disulfide  show  the  carotinoid 
nature  of  the  lipochromes.  In  general  they  favor  carotin  rather  than 
one  of  the  xanthophylls.  In  three  species,  namely,  Halma  Bucklandi, 
Halichondria  albescens  and  Leuconia  Gossei,  one  lipochrome  was 
found  showing  only  a  single  absorption  band.  The  species  Halichon- 
dria incrustans  and  Halichondria  seriata  may  contain  both  carotin 
and  xanthophylls  since  the  alcohol  remaining  after  the  carbon  disul- 
fide extraction  still  showed  carotinoid  absorption  bands.  On  the 
other  hand,  carotin  alone  may  be  the  chromolipoid  in  the  species 
Halichondria  caruncula  and  Halichondria  rosea,  whose  alcohol  ex- 
tracts were  left  practically  colorless  by  the  carbon  disulfide.  No 
information  of  a  similar  nature  is  given  for  the  species  Halichondria 
panicea,  Hymeniacidon  albescens,  Grantia  coriacea,  Halichondria  san- 
guinea  and  Pachymatisma  Johnstonia,  comprising  the  remaining 
species  examined. 


CAROTINOIDS  IN  INVERTEBRATES  171 

Summary 

Carotinoids  are  abundantly  present  in  the  invertebrates  but  they 
cannot  be  said  to  predominate  even  among  the  pigments  of  yellow 
to  red  color.  As  one  descends  the  scale  of  animal  forms  non-carotinoid 
pigments  of  yellow  to  red  hues  are  encountered  more  and  more  fre- 
quently. The  simpler  digestive  apparatus  of  the  lower  animals  does 
not  seem  to  insure  a  more  abundant  distribution  of  biologically  de- 
rived pigments. 

The  orders  of  insects  in  which  carotinoids  occur  are  butterflies,  bugs, 
beetles  and  locusts  (grasshoppers).  In  the  butterflies  it  is  the  larvae 
and  pupae  which  contain  carotinoids,  not  the  butterflies  themselves. 
Although  the  chromolipoid,  present  chiefly  in  the  haemolymph  (blood) 
of  the  larvae  and  pupae,  and  also  in  the  eggs,  has  been  known  as 
''xanthophyll"  since  the  work  of  Poulton  (1885)  there  is  evidence  to 
suggest  that  the  pigment  is  actually  carotin. 

Among  the  bugs,  the  yellow^  pigment  which  can  be  extracted  from 
green  plant  lice  (Aphids)  is  carotinoid  in  nature  but  it  is  not  known 
whether  a  single  pigment  or  a  mixture  is  concerned.  Carotin  itself 
appears  to  be  the  cause  of  the  red  color  of  the  tegument  in  the  case 
of  certain  other  species  of  bugs. 

There  can  be  no  doubt  that  the  yellow  and  orange  pigments  found 
in  the  beetles  are  often,  if  not  always,  carotinoids.  The  red  pig- 
ment, however,  belongs  to  the  carotin-like  pigments  which  conforin 
to  the  properties  of  the  so-called  carotinins  described  in  previous 
chapters. 

The  character  of  the  carotinoids  which  occur  in  many  locusts  and 
grasshoppers  is  not  known;  the  subject  deserves  further  study. 

Two  distinct  types  of  chromolipoids  are  present  in  Crustacea,  one 
characterized  by  its  red  color,  the  other  by  a  more  yellow  hue.  All 
the  older  investigations  of  the  red  pigment  agree  in  showing  that  it 
differs  in  its  general  properties  from  the  known  carotinoids  only  in 
exhibiting  one  spectroscopic  absorption  band  and  in  forming  salts 
with  alkalies  and  alkaline  earths.  However,  Verne  (1920a,  b)  has 
recently  announced  that  the  pigment  is  identical  in  every  respect 
with  carotin.  All  the  properties  of  the  yellow  pigments  so  far  ex- 
amined suggest  carotin,  rather  than  xanthophyll. 

The  red  crustacean  carotinoid  appears  to  exist  in  the  shell  of  vari- 
ous species  as  a  water-soluble  substance  of  blue,  brown,  orange  or 
green  color,  which  is  instantly  transformed  into  the  water-insoluble 


172  CAROTINOIDS  AND  RELATED  PIGMENTS 

red  carotinoid  by  heat,  acids,  alcohol,  ether,  etc.  The  author  sug- 
gests a  colloidal  explanation  for  the  hitherto  inexplicable  relations 
between  these  apparently  water-soluble  chromogens  and  the  red 
pigment. 

The  same  red  and  yellow  carotinoid-like  pigments  found  in  Crus- 
tacea appear  to  be  the  cause  of  like  colorations  among  the  echino- 
derms  (starfish,  brittle-stars,  sea-urchins,  etc.).  In  addition,  red, 
green,  blue  and  violet  lipochromogens  are  also  encountered. 

The  shells  of  snails  apparently  may  be  colored  by  the  red  caroti- 
noid-like pigment  already  described,  and  the  digestive  organs  of  these 
animals  as  well  as  some  molluscs  may  contain  a  pigment  which  is 
either  carotin  or  xanthophyll  or  a  modification  of  one  of  these 
carotinoids. 

The  brilliant  colors  encountered  among  marine  and  fresh  water 
worms  are  due  in  part  to  carotin  or  related  pigments.  Similar  colors 
among  the  sea-anemones,  corals,  jelly-fish  and  related  animals,  how- 
ever, do  not  appear  to  involve  the  carotinoids. 

The  yellow,  orange  and  red  colors  of  sponges  have  been  found  to 
be  due  almost  exclusively  to  lipochromes  in  the  broad  sense.  The 
yellow  and  orange  pigments  are  undoubtedly  true  carotinoids,  and 
the  red  pigment  is  the  carotin-like  substance,  showing  one  absorp- 
tion band,  which  is  so  widely  distributed  among  lower  forms  of  ani- 
mal life.  The  presence  of  both  carotin  and  xanthophylls  is  indicated 
among  the  true  carotinoids. 


i 


Chapter  VI 

Chemical  Relations  between  Plant  and  Animal 
Carotinoids 

It  is  a  chemical  axiom,  so  to  speak,  that  the  final  proof  of  the 
identity  of  like  chemical  compounds  must  be  furnished  by  a  chemi- 
cal analysis  of  the  purified  substances,  together  with  complete  cor- 
respondence in  all  known  chemical  and  physical  properties.  It  has 
been  stated  repeatedly  in  the  preceding  chapters  that  evidence  has 
been  presented  which  shows  the  character  of  the  chemical  relationship 
between  plant  and  animal  carotinoids.  It  is  desired  to  consider  this 
evidence  in  more  detail  in  this  chapter. 

Egg  yolk  xanthophyll.  It  is  not  necessary  to  present  again  the 
observations  showing  the  close  relationship  between  the  pigment  of 
the  yolk  of  the  hen's  egg  and  the  plant  chromolipoids,  which  was 
known  to  numerous  workers  through  the  macroscopic  examination  of 
the  pigment.  Reference  may  be  made,  however,  to  the  spectroscopic 
studies  of  Schunck  (1903)  who  first  showed  the  correspondence  be- 
tween the  egg  yolk  pigment  and  one  of  the  groups  of  plant  carotinoids. 
Schunk's  results,  secured  largely  by  a  photographic  study  of  the 
absorption  spectra  of  the  pigments  separated  by  inadequate,  and 
unfortunately  by  inaccurate  means,  were  obtained  before  carotin  and 
xanthophylls  were  established  as  chemical  entities.  This  method  was 
described  in  Chapter  II.  It  could  not  have  insured  the  freedom  of 
the  "xanthophylls"  from  admixture  with  carotin.  Nevertheless 
Schunck  was  careful  to  distinguish  between  xanthophylls  and  "chry- 
sophyll,"  which  he  recognized  as  probably  identical  with  carotin. 
The  spectrophotographs  show  this  very  clearly  so  that  the  spectro- 
scopic relations  between  one  of  Schunck's  flower  xanthophylls  (his 
so-called  L.  xanthophyll)  and  the  egg  yolk  pigment  rightly  deserve 
credit  for  being  the  first  to  show  the  xanthophyll  character  of  this 
animal  chromolipoid. 

Crystals  of  egg  yolk  pigment  are  stated  by  Willstatter  and  Escher 
(1912)  to  have  been  observed  first  by  Kiihne  (1882).    It  was  stated 

173 


174  CAROTINOIDS  AND  RELATED  PIGMENTS 

in  Chapter  III  that  Kiihne  (1878)  had  previously  decided,  partly  on 
spectroscopic  grounds,  that  the  egg  yolk  pigment  could  not  be  identi- 
cal with  the  so-called  lutein  in  the  corpus  luteum.  It  remained, 
however,  for  Willstatter  and  Escher  to  attempt  the  isolation  of  the 
pigment  in  sufficient  quantity  for  chemical  analysis.  This  proved  to 
be  a  rather  difficult  task  and  involved  working  with  large  quantities 
of  material.  Starting  with  the  yolks  of  6,000  eggs,  which  weighed 
110  kg.,  only  4  grams  of  crude  crystalline  pigment  was  obtained. 
The  method  of  isolation  will  be  described  in  Chapter  VIII. 

The  crude  egg  yolk  pigment  was  purified  first  by  repeated  crystal- 
lization from  hot  methyl  alcohol.  About  250  cc.  of  boiling  alcohol 
were  required  to  dissolve  0.25  grams  of  the  crude  product,  from  which 
approximately  0.16  grams  of  crystals  came  down  on  standing  for 
some  hours.  It  is  stated  that  it  was  also  found  possible  to  obtain 
crystals  showing  a  constant  melting  point  by  dissolving  the  crude 
crystals  in  carbon  disulfide  and  recrystallizing  from  this  solvent.  The 
chemical  studies  on  the  purified  substance  showed  the  following  aver- 
age results.  For  comparison  similar  data  are  shown  for  the  plant 
xanthophyll  isolated  by  Willstatter  and  Mieg  (1907). 

Plant  Egg  yolk 
xanthophyll       xanthophyll 
CHaOH      of      crystallization,      calculated      for 

C40H56O2,    CH3OH =  per  cent                   5.33  5.33 

CH3OH    found percent                  4.76  4.35 

Molecular     weight      in      CHCI3,      ebulloscopic 

method,  calculated  for  C40H56O2 568.4  568.4 

Molecular  weight  found 512.  640. 

Elementary    analysis ;    percentage    compositions  C  =      84.44  84.44 

required  for  formula  C40H56O2 ^  H  =        9.93  9.93 

Elementary  a^nalysis  found ; 5  C  =      84.22  83.58 

I H  =        9.92  10.13 

Melting-point    (corrected) 173.5  — 174.5°     195  —  196°C. 

An  examination  of  these  data  shows  that  the  plant  xanthophyll 
analyzed  by  Willstatter  and  Mieg  showed  excellent  agreement,  at 
least  in  chemical  composition,  with  the  theoretical  values.  The  re- 
sults of  the  analyses  of  the  egg  yolk  pigment,  however,  can  only  be 
regarded  as  approximations,  at  best.  The  molecular  weight  deter- 
mination is  also  very  unsatisfactory.  Willstatter  and  Escher  explain 
the  low  figure  for  the  content  of  methyl  alcohol  of  crystallization  on 
the  grounds  of  a  possible  oxidation  of  the  pigment  during  the  process 
of  removal  of  the  alcohol,  which  required  a  period  of  about  10  days 
over  phosphorus  pentoxide.  The  explanation  does  not  seem  entirely 
satisfactory,  however,  in  view  of  the  statement  that  this  process  took 


CHEMICAL  RELATIONS  BETWEEN  CAROTINOIDS    175 

place  in  a  high  vacuum.  No  similar  explanation  is  offered  for  the 
divergence  of  the  molecular  weight  and  elementary  composition  deter- 
minations from  the  theoretical  values.  It  is  not  apparent  from  the 
description  given  that  any  less  care  was  taken  in  purifying  the  crys- 
tals for  these  analyses  than  was  taken  by  Willstatter  and  Mieg  in 
preparing  the  plant  xanthophyll  for  a  like  purpose. 

When  these  results  are  viewed  from  a  strictly  chemical  standpoint 
it  is  difficult  to  escape  the  conclusion  that  the  purest  preparations 
were  not  free  from  admixture  with  a  substance  of  high  molecular 
weight  which  is  lower  in  carbon  and  higher  in  hydrogen  than  the 
xanthophyll.  This  conclusion  is  supported  by  Willstatter  and 
Escher's  own  statement  that  the  4  grams  of  crude  pigment  contained 
a  large  proportion  of  wax-like  material  which  had  solubility  prop- 
erties similar  to  the  pigment.  On  the  other  hand,  it  is  hardly  pos- 
sible that  the  high  melting  point  of  the  egg  yolk  pigment  in  compari- 
son with  the  plant  xanthophyll,  which  led  Willstatter  and  Escher  to 
call  the  pigment  xanthophyll  "b,"  was  due  to  the  impossibility  of 
removing  the  unknown  impurity.  The  presence  of  a  wax  of  high 
molecular  weight  would  undoubtedly  alter  the  melting  point  of  the 
pure  pigment  but  would  lower,  rather  than  raise  it.  There  is  cer- 
tainly no  evidence  that  the  preparations  used  for  the  melting  point 
determinations  were  of  any  higher  purity  than  those  used  for  the 
elementary  analyses. 

The  other  chemical  properties  of  the  egg  yolk  xanthophyll  leave  no 
doubt  as  to  its  identity  in  these  respects  with  the  plant  xanthophyll. 
The  actual  solubility  of  the  two  pigments  in  various  solvents  is  iden- 
tical. The  crystalline  form  from  various  solvents  agrees  perfectly. 
Both  pigments  form  a  violet  colored  crystalline  iodide  (showing  that 
Kiihne's  failure  to  obtain  the  blue  iodine  lipochrome  reaction  was  not 
due  to  any  peculiar  characteristic  of  the  pigment).  The  phase  test 
applied  to  the  purified  pigment  shows  complete  correspondence  with 
the  xanthophyll  group  of  carotinoids,  the  pigment  being  practically 
quantitatively  hypophasic  between  petroleum  ether  and  80-90  per 
cent  ethyl  or  methyl  alcohol.  Finally,  the  spectroscopic  absorption 
bands  of  the  two  pigments  are  identical  in  every  respect  when  meas- 
ured under  the  same  conditions. 

As  pointed  out  in  Chapter  IV  there  is  also  a  biological  basis  for 
rejecting  Willstatter  and  Escher's  conclusion  that  the  egg  yolk  xan- 
thophyll is  an  isomer  of  plant  xanthophyll,  unless  it  be  assumed  that 
the  plant  xanthophyll  from  which  the  egg  yolk  pigment  is  derived  is 


176  CAROTINOIDS  AND  RELATED  PIGMENTS 

modified  in  the  animal  body.  The  hypothesis  was  also  advanced  that 
the  egg  yolk  xanthophyll  may  be  an  individual  member  of  the  xantho- 
phyll  group  of  carotinoids,  which  the  digestive  and  assimilative  organs 
of  the  hen  have  the  ability  to  select  from  the  group  of  carotinoids  pre- 
sented to  them  in  the  food.  Considered  solely  from  a  chemical  basis, 
however,  it  is  indeed  an  extraordinarily  closely  related  isomer  which 
shows  such  complete  correspondence  in  all  its  other  properties,  both 
chemical  and  physical,  with  the  single  exception  of  the  melting  point. 
Lycopin,  the  red  isomer  of  carotin,  found  especially  in  the  tomato, 
has  the  same  melting  point  and  chemical  composition  as  carotin,  but 
differs  from  it  in  a  number  of  chemical  properties,  such  as  color, 
absorption  spectrum,  solubilities,  etc.  Therefore,  from  this  point  of 
view,  also,  it  seems  unlikely  that  the  alleged  isomerism  of  the  egg 
yolk  xanthophyll  actually  exists. 

Serono  and  Palozzi  (1911)  claimed  to  have  isolated  the  lutein  of 
egg  yolk  by  a  very  different  method.  Egg  yolk  was  extracted  with 
95  per  cent  alcohol,  the  extract  evaporated  in  vacuum,  and  the  residue 
treated  with  acetone.  This  extract  is  stated  to  have  contained  mostly 
"lutein,"  a  little  cholesterol  and  traces  of  lecithin.  The  lutein  was 
obtained  in  crystalline  form  from  this  solution  by  precipitation  from 
boiling  acetone.  The  crystals  thus  secured  are  described  as  white  to 
pale  yellow  radiating  clusters  with  a  few  crystalline  lamella  with  a 
blue  fluorescence,  which  turn  yellow  almost  instantly  in  the  air  and 
become  more  and  more  colored  until  a  deep  red  is  reached.  The 
analyses  of  this  lutein  indicated  a  mixture  of  cholesterol,  fat  and 
cholesterol  esters  of  oleic  and  palmitic  acids.  Egg  yolk  is  stated  to 
contain  about  4.04  to  4.17  per  cent  of  this  pigment. 

With  the  above  observations  as  a  basis  it  is  not  surprising  that 
Serono  (1912)  vigorously  attacked  Willstatter  and  Escher's  work 
showing  the  xanthophyll  nature  of  the  egg  yolk  pigment.  It  is  obvi- 
ous, of  course,  that  Serono's  lutein  cannot  be  the  true  egg  yolk  pig- 
ment. At  the  same  time  the  poor  correspondence  of  the  egg  yolk 
xanthopyhll  with  the  plant  pigment  with  respect  to  the  melting  point 
and  elementary  composition  naturally  offered  splendid  points  of  at- 
tack. Accordingly  Serono's  assertion  is  quite  incontrovertible  that  the 
carbon  content  found  for  the  egg  yolk  pigment  by  Willstatter  and 
Escher  (83.58  per  cent)  corresponds  better  with  the  carbon  content 
of  an  oleic  acid  ester  of  cholesterol  (83.33  per  cent)  than  it  does  with 
the  carbon  content  of  carotin  dioxide  (84.44  per  cent) .  Although  one 
would  hardly  be  tempted  to  accept  Serono's  conclusions  regarding  a 


CHEMICAL  RELATIONS  BETWEEN  CAROTINOIDS    111 

cholesterol  ester  constitution  for  the  egg  yolk  pigment,  it  must  be 
admitted,  nevertheless,  that  if  it  were  not  for  the  complete  corre- 
spondence of  the  general  chemical  properties  of  the  egg  yolk  pigment 
with  plant  xanthophyll,  it  would  not  be  possible  to  decide  on  chemi- 
cal grounds,  from  the  evidence  submitted,  that  the  two  substances 
are  identical  or  even  isomers. 

Corpus  luteum  carotin.  It  was  pointed  out  in  a  previous  chapter 
that  the  pigment  in  the  corpus  luteum  tissue  on  the  ovaries  of  the  cow 
was  one  of  the  first  to  attract  the  attention  of  those  interested  in  dis- 
covering the  nature  of  animal  pigments.  It  was  certainly  the  first 
animal  carotinoid  to  be  obtained  in  crystalline  form  if  one  is  to  accept 
the  early  work  of  Piccolo  and  Lieben  (1866)  and  Holm  (1867). 
Although  a  number  of  later  workers  described  the  chemical  prop- 
erties of  the  pigment,  as  was  shown  in  Chapter  IV,  Willstatter  an^ 
Escher  (1912)  are  to  be  credited  with  first  stating  the  exact  chemical 
relationship  of  the  corpus  luteum  pigment  to  plant  carotin.  Their 
work  was  not  the  first  to  show  a  connection  between  animal  coloring 
matter  and  plant  carotin;  nor  were  they  the  first  to  use  the  name 
carotin  for  an  animal  pigment.  It  will  be  recalled  that  Zopf  used 
the  name  carotin  in  the  form  of  "eucarotin,"  "di-carotin,"  "mono- 
carotin,"  "carotinin,"  etc.,  for  various  animal  pigments  studied  by 
him.  He  certainly  recognized  the  relation  between  the  vegetable  and 
animal  "carotins,"  to  which  he  gave  the  various  designations  men- 
tioned. It  is  apparent,  however,  that  the  name  itself  was  a  collective 
name  in  Zopf's  mind  and  that  both  carotin  and  xanthophylls,  in  the 
sense  we  now  know  them,  were  included.  For  example,  Gerlach 
(1892)  working  under  Zopf's  direction,  refers  to  the  egg  yolk  pigment 
as  di-carotin.  On  the  other  hand  Physalix  (1894)  actually  had 
Arnaud's  carrot  carotin  in  mind,  as  his  paper  shows,  when  he  assigned 
the  name  carotin  to  the  pigment  which  he  isolated  from  the  red  tegu- 
ment of  the  bug  Pyrrhocoris  apterus.  The  insect  carotin  was  not, 
however,  analyzed. 

It  was  no  doubt  the  development  of  the  Kraus  method  for  separat- 
ing the  plant  carotinoids  into  specific  carotin  and  xanthophyll  groups, 
in  which  Tswett  and  Willstatter  played  a  dominant  part,  that  led 
Willstatter  to  examine  certain  of  the  common  animal  lipochromes  by 
this  procedure.  The  discovery  of  the  xanthophyll  character  of  the 
egg  yolk  pigment  by  this  means  naturally  prompted  the  study  of  the 
corpus  luteum  pigment,  since  the  observations  of  the  early  workers 
had  shown  that  it  could  be  obtained  in  crystalline  form  without  dif- 


178  CAROTINOIDS  AND  RELATED  PIGMENTS 

ficulty.  Willstatter  and  Escher  announced  their  discovery  of  the 
carotin  nature  of  the  corpus  luteum  pigment  in  connection  with  their 
studies  on  egg  yolk  xanthophyll.  The  details  of  the  corpus  luteum 
work  were  later  published  by  Escher  (1913). 

The  relatively  small  size  of  corpora  lutea  tissue  naturally  required 
the  collection  of  ovaries  on  a  large  scale.  It  was  fortunately  found 
possible  to  preserve  the  ovaries  for  many  months  under  60  per  cent 
alcohol  without  impairment  of  the  pigment  (preservation  in  dilute 
formalin  prevented  the  isolation  of  crystals) ,  thus  making  it  feasible 
to  collect  the  material  in  large  slaughter  houses  over  an  extended 
period  of  time.  After  146  kgs.  (about  10,000  ovaries)  has  been  col- 
lected from  cows  and  sheep,  the  tissue  was  hashed  in  10  kg.  lots, 
further  dehydrated  with  95  per  cent  alcohol,  and  then  shaken  in  the 
cold  with  petroleum  ether  (b.  p.  50°-70°  C.)  for  several  hours. 
This  ejffected  an  almost  complete  extraction  of  pigment.  This  extract 
(about  3  liters  for  each  10  kg.  of  ovaries)  was  then  washed  seven  suc- 
cessive times  with  one-sixth  volume  of  90  per  cent  methyl  alcohol, 
the  alcohol  removed  by  washing  four  times  with  one-third  volume  of 
water,  the  extract  freed  from  water  by  shaking  with  anhydrous  sodium 
sulfate,  filtered  and  concentrated  to  a  syrup  in  vacuum.  On  adding 
six  to  ten  volumes  of  absolute  ethyl  alcohol  and  cooling  in  an  ice-salt 
bath  nearly  all  fatty  impurities  were  precipitated.  These  were  sepa- 
rated quickly  by  means  of  a  cloth  filter,  for  in  a  short  time  crystalliza- 
tion of  the  pigment  began  and  continued  for  several  hours,  at  ice-box 
temperature.  Some  very  large  (1-2  mm.  long),  beautiful  crystals 
were  obtained.  Purification  was  secured  by  filtering  and  washing 
with  a  mixture  of  equal  parts  petroleum  ether  and  absolute  alcohol. 
Only  0.45  grams  of  pigment  in  all  were  secured  in  this  way  from  the 
146  kgs.  of  ovaries. 

The  crude  ovarian  pigment  proved  to  be  remarkably  pure  as  judged. 
from  the  microscopic  examination  and  melting  point  of  the  crystals. 
For  the  elementary  analyses,  however,  the  pigment  was  recrystal- 
lized  first  from  alcohol,  again  by  addition  of  an  excess  of  absolute 
alcohol  to  a  concentrated  carbon  disulfide  solution,  and  finally  from 
pure  petroleum  ether  (sp.  g.  0.64-.66).  The  elementary  composition 
showed  the  pigment  to  be  a  hydrocarbon.  The  average  of  four  closely 
concordant  analyses  carried  out  for  Escher  by  Fisher  and  Sonnenfeld 
under  Willstatter's  direction,  using  Preyl's  micro-method,  showed  89.55 
per  cent  carbon  and  10.66  per  cent  hydrogen.  This  corresponds  even 
more  closely  to  the  calculated  composition  of  the  compound  C40H66 


CHEMICAL  RELATIONS  BETWEEN  CAROTINOIDS    179 

than  Willstatter  and  Mieg  obtained  in  their  analyses  of  the  carotin 
from  either  green  leaves  or  carrots.  Although  Escher  was  unable  to 
carry  out  any  molecular  weight  determinations  because  of  lack  of 
material,  there  can  be  no  doubt  from  these  data  that  the  ovarian 
carotin  is  identical  in  composition  with  the  carotin  of  vegetable  origin. 

Escher's  observations  of  the  melting-point  of  ovarian,  carrot  and 
leaf  carotins  are  of  interest.  By  heating  crystalline  preparations  from 
each  source  together  in  the  same  bath  using  a  shortened  thermometer 
he  found  that  they  all  melted  at  exactly  the  same  temperature,  but 
that  this  temperature  was  about  175°  C.  (corrected).  This  tem- 
perature is  appreciably  higher  than  the  figure  reported  by  Willstatter 
and  Mieg  for  the  carotins  of  plant  origin,  namely,  167.5°-168°  C, 
which  corresponded  with  the  earliest  observations  of  Arnaud,  Kohl 
and  others.  Escher  explained  his  results  on  the  grounds  that  con- 
siderable variation  in  melting  point  can  be  obtained  by  modifying  the 
method  of  heating,  so  that  in  reality  the  melting  point  of  crystals  of 
different  carotinoids  should  be  determined  under  comparative  con- 
ditions to  secure  comparable  results.  These  results  may  indicate, 
therefore,  that  the  slight  difference  in  the  melting  point  of  carotin 
and  xanthophyll  crystals  may  not  be  of  great  importance  in  distin- 
guishing the  two  forms  of  carotinoid. 

The  general  properties  of  the  ovarian  carotin  also  correspond  exactly 
with  the  carotin  from  carrots  and  leaves.  The  crystalline  form; 
solubility  of  the  crystals  in  various  solvents;  response  to  the  phase 
test  (being  practically  quantitatively  epiphasic  between  petroleum 
ether  and  80-90  per  cent  alcohol) ;  position  of  the  absorption  bands; 
formation  of  a  crystalline  tri-iodide  (C^oHgJs)  in  carbon  disulfide 
solution,  melting  at  133.5°-135°  C,  and  showing  40.6  per  cent  iodine 
(calculated  value  41.5  per  cent) ;  and  its  ready  oxidation  to  a  grayish- 
yellow  powder,  soluble  in  alcohol  and  containing  36  per  cent  oxygen, 
all  corresponded  exactly  with  the  properties  of  carotin  from  leaves 
and  carrots.  There  can  be  no  doubt  whatever  that  the  carotin  from 
the  corpus  luteum  of  the  •  cow  is  identical  with  the  carotin  so  widely 
distributed  in  the  vegetable  kingdom. 

Crustacean  carotin.  As  stated  in  Chapter  V  in  connection  with  the 
chromolipoids  of  Crustacea,  Verne  (1920)  has  recently  stated  that  the 
red  pigment  in  the  hypodermis  of  the  higher  Crustacea,  such  as  lob- 
sters, crabs  and  crayfish,  is  identical  with  carrot  carotin.  The  analyti- 
cal data  are  not  presented.  In  support  of  his  conclusion,  however, 
Verne  makes,  in  substance,  the  following  assertions.     The  pigment 


180  CAROTINOIDS  AND  RELATED  PIGMENTS 

was  isolated  by  the  methods  of  Arnaud  and  Escher.  The  crystals 
melted  at  168°  C,  and  gave  an  iodide  of  definite  composition.  The 
elementary  analyses  of  a  large  number  of  samples  obtained  from 
Crustacea  of  various  kinds  showed  the  pigment  to  be  a  hydrocarbon  in 
which  the  ratio  of  C :  H  =  5 : 7.  The  molecular  weight  determination 
performed  on  the  iodides  by  the  ebulloscopic  method  showed  that  the 
carotin  of  Crustacea  is  the  same  as  that  of  vegetables,  with  the  form- 
ula C^oHgg.  Among  its  most  significant  reactions  was  the  formation 
of  a  violet-brown  iodide.  The  pigment  exhibited  the  same  spectro- 
scopic absorption  bands  as  vegetable  carotin.  It  was  not  attacked 
by  alkalis  and  oxidized  with  great  ease. 

These  statements  are  certainly  sufiicient  to  establish  the  carotin 
identity  of  the  crustacean  pigment.  It  is  to  be  hoped,  however,  that 
the  details  of  this  study  will  soon  be  made  available.  Certain  of  the 
points  mentioned  in  connection  with  the  general  properties  of  the  pig- 
ment are  quite  at  variance  with  the  findings  of  numerous  previous 
investigators  who  were  presumably  studying  the  same  crustacean  pig- 
ment. Either  Verne  was  working  with  a  different  pigment,  or  the 
methods  used  by  previous  investigators  were  decidedly  at  fault.  It  is 
important  that  these  divergencies  be  explained. 

Summary 

A  chemical  relationship  between  an  animal  chromolipoid  and  a 
specific  plant  carotinoid  was  shown  for  the  first  time  by  Schunck's 
(1903)  comparative  spectroscopic  studies  of  flower  "xanthophylls" 
and  the  yellow  pigment  of  the  egg  yolk  and  blood  serum  of  fowls. 

Chemical  studies  of  crystals  of  egg  yolk  pigment  by  Willstatter  and 
Escher  (1912)  showed  a  complete  correspondence  with  plant  xantho- 
phyll  in  all  properties  except  melting  point,  but  the  results  of  the  ele- 
mentary analyses  and  molecular  weight  determinations  of  the  egg 
yolk  pigment  can  only  be  regarded  as  approximations  to  the  theoreti- 
cal values  for  a  substance  with  the  formula  C^oHgeOg.  A  considera- 
tion of  these  divergencies  in  the  light  of  the  biological  relationship 
between  egg  yolk  pigment  and  plant  xanthophyll  leads  to  doubts 
regarding  the  alleged  isomerism  of  the  two  pigments. 

A  possible  chemical  relationship  between  certain  animal  chromo- 
lipoids  and  plant  carotin  was  recognized  by  several  workers  before 
Escher  (1913)  definitely  established  the  chemical  identity  of  the  cor- 
pus luteum  pigment  (of  the  cow)  with  plant  carotin. 


I 


CHEMICAL  RELATIONS  BETWEEN  CAROTINOIDS    181 

Escher's  study  of  the  comparative  melting  points  of  ovarian,  carrot 
and  leaf  carotin  shows  that  slight  differences  between  the  melting 
point  of  different  carotinoids  are  not  significant  unless  the  determi- 
nations are  carried  out  simultaneously. 

The  recent  work  of  Verne  (1920)  on  the  identity  of  the  red  crus- 
tacean chromolipoid  with  plant  carotin  throws  doubt  on  the  results  of 
numerous  previous  workers  which  indicate  that  the  pigments  are 
related  but  not  identical. 


Chapter  VII 

Biological  Relations  between  Plant  and  Animal 
Carotinoids 

The  origin  of  color  in  animals,  when  considered  by  and  large,  has 
been,  until  recently,  practically  an  unexplored  field.  So  far  as  the 
colors  which  might  be  due  to  carotinoids  or  related  pigments  are  con- 
cerned no  systematic  study  of  their  possible  biological  relationship  to 
plant  pigments  of  similar  color  was  undertaken  previous  to  the  inves- 
tigations by  Palmer  and  Eckles,  published  in  1914.  A  few  close 
students  of  the  subject  have,  indeed,  suggested  such  a  relationship  in 
isolated  cases,  which  are  mentioned  below,  and  there  are  also  certain 
isolated  observations  which  support  the  idea.  It  is  a  striking  fact, 
however,  that  so  little  experimental  work  had  been  done  in  this  field 
that  even  the  chemical  identification  of  certain  of  the  animal  lipo- 
chromes  with  plant  carotinoids  by  Willstatter  and  Escher  (1912) 
and  by  Escher  (1913)  apparently  raised  no  query  in  their  minds  as 
to  their  possible  origin  from  the  plant  pigments.  For  example, 
Escher,  in  concluding  the  paper  on  corpus  luteum  carotin  remarks, 
"What  this  unsaturated  terpene  hydrocarbon,  carotin,  which  is  so 
widely  distributed  in  the  plant  world,  is  doing  in  such  an  important 
gland  as  the  corpus  luteum,  can  not  even  be  conjectured  at  present. 
— there  is  nothing  in  the  literature  to  establish  whether  it  is  a  sub- 
stance produced  from  one  of  the  specific  gland  cells,  or  is  only  a 
pigment  which  has  been  resorbed  by  the  cells  from  the  blood  ex- 
travates."  It  is  clear,  also,  that  Escher  saw  no  biological  relation- 
ship between  the  xanthophyll  of  the  egg  yolk  and  plant  xanthophylls, 
for  he  expresses  the  view  that,  "the  oxygen-containing  lutein 
(C40II56O2)  in  the  yolk  of  eggs  plays  the  part  of  an  atavistic  plant 
respiratory  pigment  for  the  formation  of  hemoglobin  in  the  embryo." 

It  is  true  that  when  Fischer  and  Rose  (1913)  isolated  carotin  from 
the  gall  stones  of  cattle  they  were  unable  to  agree  with  Escher's  con- 
clusion and  suggested,  rather,  that  the  carotin  in  the  cow's  body 
probably  comes  from  the  food.    In  reality,  however,  Escher's  general 

182 


( 


BIOLOGICAL  RELATIONS  BETWEEN  CAROTINOIDS    183 

view  that  the  animal  lipochromes  are  of  animal  origin  coincides  with 
Krukenberg's    (1886)    conclusion   in  the   summary   of   his   extensive 
chromolipoid  studies,  where  it  is  stated  that,  "they    (i.e.,  the  lipo- 
chromes) originate  in  most  cases  from  fat-like  substances."    A  more 
specific  instance  of  the  same  general  conclusion  reached  by  one  of  the 
foremost  earlier  workers  on  animal  lipochromes  is  found  in  the  paper 
by  Zopf   (1893a)   describing  the  yellow  carotin-like  pigment  in  the 
little  fresh-water  crustacean,  Diaptomus  bacillifer.     Zopf  states,  *'I 
could  mention  an  objection  which  could  be  raised  against  the  two- 
banded  yellow  carotin  found  in  these  Crustacea.     One  could  say  that 
it  is  not  produced  by  the  organs  of  the  crab  but  perhaps  comes  from 
the  chlorophyll-containing  algae  which  serve  as  their  food,  and  which 
contain  a  two-banded  yellow  carotin.    However,  this  can  not  be  true 
because  the  animals  with  which  I  worked  were  preserved  in  alcohol 
and  no  trace  of  chlorophyll  was  extracted  which  would  have  been  the 
case  had  algse  been  present  in  their  digestive  tract." — "The  antennae 
of  Diaptomus  Castor  Jurine  is  colored  exclusively  by  diaptomin  while 
the  body  cavity  contains  fat  masses  of  purest  yellow.    I  believe  that 
the  yellow  carotin  is  produced  by  these  animals  just  like  the  diap- 
tomin."    It  is  clear  from  these  citations  that  Zopf  saw  no  evidence 
of  a  biological  relationship  between  plant   and  animal   carotinoids. 
On  the  other  hand  Poulton  (1885)  believed  that  yellow  pigment  in 
caterpillars  was  derived  from  the  "xanthophyll"  (carotinoids)  of  the 
food  and  the  green  color  from  chlorophyll.    Poulton  later  (1893)  sub- 
mitted his  experimental  proof  of  the  derivation  of  chlorophyll  by  the 
caterpillar  from  its  food,  an  experiment  which  is  classic  so  far  as  the 
demonstration  of  derived  pigments  in  animal  colorations  is  concerned. 
Goode  (1890)  made  a  particularly  interesting  observation,  which  can 
hardly  be  classed  as  an  experiment,  but  which  verifies  the  probability 
of  a  biological  relationship  between  plant  and  animal  pigments  in 
another  species.    To  quote  directly  from  his  paper:  "On  certain  ledges 
along   the   New   England   coast  the   rocks   are   covered  with    dense 
growths  of  scarlet  and  crimson  seaweeds.     The  codfish,  the  cunner, 
the  sea  raven,  the  rock-eel,  and  the  wrymouth,  which  inhabit  these 
brilliant  groves,  are  all  colored  to  match  their  surroundings;  the  cod, 
which  has  naturally  the  lightest  color,  being  most  brilliant  in  its 
scarlet  hues,  while  the  others,  whose  skins  have  a  larger  original  sup- 
ply of  black,  have  deeper  tints  of  dark  red  and  ruddy  brown." — "It 
has  occurred  to  me  that  the  material  for  the  pigmentary  secretion  is 
probably  derived  indirectly  from  the  algae,  for,  though  the  species 


184  CAROTINOIDS  AND  RELATED  PIGMENTS 

referred  to  do  not  feed  upon  these  plants,  they  devour  in  immense 
quantities  the  invertebrate  animals  inhabiting  the  same  region,  many 
of  which  are  likewise  deeply  tinged  with  red.  Possibly  the  blacks  and 
greens  which  prevail  among  the  inhabitants  of  other  colored  bottoms 
are  likewise  dependent  upon  coloring  matter  which  is  absorbed  with 
the  food.  Giinther  believes  that  the  pink  color  in  the  flesh  of  the 
salmon  is  due  to  the  absorption  of  the  coloring  matter  of  the  crus- 
taceans they  feed  upon." 

Miss  Newbigin,  both  in  her  papers  (1898)  and  her  Monograph 
(1898)  takes  a  somewhat  intermediate  position  on  the  question  of 
derived  animal  pigments.  Regarding  insects,  she  accepts  Poulton's 
results  but  qualifies  them  by  stating  that,  "At  the  same  time  there  is 
no  apparent  reason  why  insects  should  not  themselves  produce  lipo- 
chromes,  and  why  such  lipochromes  should  not  occur  in  the  cuticle 
as  in  the  Crustacea."  With  reference  to  the  carotinoids  in  birds,  she 
states  with  more  conviction,  that  "although  there  are  several  instances 
described  of  birds  whose  colors  can  be  heightened  or  altered  by  the 
employment  of  special  kinds  of  food,  there  is  at  present  no  reason  to 
doubt  that  under  ordinary  circumstances  the  lipochromes  of  birds 
are  self-produced  and  not  derived." 

Miss  Newbigin  gives  a  more  extensive  presentation  of  her  views  on 
this  subject  in  her  paper  on  salmon  pigments  (1898).  It  will  be  re- 
called that  she  found  a  red  lipochrome  and  a  yellow  pigment  which 
she  could  not  identify  as  a  true  lipochrome  in  the  muscle  and  ovaries 
of  this  fish.  In  discussing  these  findings  she  states,  "The  most  obvious 
explanation  is  that  the  pigments  of  the  salmon  are  derived  directly 
from  its  food.  ...  At  first  sight  the  suggestion  has  much  to  recom- 
mend it.  .  .  .  There  are,  however,  some  difiiculties  in  the  way  of  the 
acceptance  of  this  suggestion.  In  the  first  place,  the  salmon  seems  to 
feed  chiefly  on  haddock,  herring,  and  similar  fish,  so  that  the  transfer 
of  pigment  can  hardly  be  direct.  The  herring,  however,  feeds  habitu- 
ally on  small  Crustacea,  so  that  it  might  be  said  that  the  pigments  of 
the  salmon  are  obtained  indirectly  from  herring  which  forms  its  food." 
Miss  Newbigin,  however,  was  unable  to  find  the  red  pigment  in  her- 
ring, but  did  find  a  small  amount  of  the  yellow  pigment  in  the  viscera 
and  muscles.  In  support  of  the  general  proposition  of  animal  lipo- 
chromes being  derived  from  the  food  Poulton's  experiments  are  first 
cited  and  then  the  fact  that,  "it  is  not  uncommon  to  find  the  fat  of 
sheep  (f)  ^  and  cows  dyed  a  deep  yellow  color.    According  to  some 

*  Question  by  author. 


BIOLOGICAL  RELATIONS  BETWEEN  CAROTINOIDS    185 

authorities,  this  occurs  quite  sporadically  without  known  cause,  while 
according  to  others  special  foods,  notably  maize,  are  the  important 
agents."  Miss  Newbigin  then  states  that  she  secured  the  lipochrome 
color  reactions  with  the  maize  pigment  but  not  with  the  pigment  from 
yellow  fat.  "In  other  respects,  in  tint,  in  solubility,  and  so  on,  the 
pigments  closely  resemble  each  other.  This  fact,  taken  in  combination 
with  Mr.  Poulton's  experiments,  seems  to  me  at  least  to  prove  the 
possibility  of  the  transference  of  these  pigments  from  one  organism 
to  another,  and  therefore  to  suggest  such  an  origin  for  the  yellow  pig- 
ment of  the  salmon." 

On  further  consideration  Miss  Newbigin  concluded  that  the  deriva- 
tion of  yellow  pigment  from  the  food  could  not  be  very  general,  other- 
wise pigmented  fat  would  be  universally  found  in  herbivorous  animals, 
which  Miss  Newbigin  knew  is  not  the  case.  Her  explanation  of  the 
phenomenon  which  she  believed  to  be  peculiar  to  caterpillars,  salmon 
and  domesticated  cattle  was  that  they  ingested  more  colored  fat  in 
their  food  than  they  required  with  the  result  that,  "fat  colored  with 
the  pigment  in  more  or  less  modified  condition  is  deposited  in  certain 
of  the  tissues." 

Miss  Newbigin's  views  are  quoted  at  some  length  because  they  not 
only  have  a  direct  bearing  on  some  of  the  experiments  of  Palmer  and 
Eckles,  but  they  are  the  most  definite  of  any  of  the  earlier  views  on 
the  subject  of  a  possible  general  origin  of  animal  lipochromes  from 
plants.  Although  Miss  Newbigin  decided  against  any  such  general 
relationship  between  plant  and  animal  pigments  the  writer  has  found 
several  isolated  observations  which  support  the  idea  when  viewed  in 
the  light  of  our  present  knowledge.    These  may  be  mentioned  briefly. 

Schneidet  (1799)  observed  a  great  many  years  ago  that  the  toad, 
Bufo  viridis,  loses  its  green  color  on  wintering  in  the  earth.  Von 
Wittich  (1854)  noted  that  the  frog,  Rana  esculenta,  took  on  a  grayish- 
brown  instead  of  its  usual  green  color  after  long  fasting  and  that  this 
was  accompanied  by  a  disappearance  of  the  yellow  cells  in  the  tegu- 
ment. The  disappearance  of  the  lipochromes  from  the  skin  of  fasting 
frogs  was  confirmed  by  Hering  and  Hoyer  (1869),  who  also  noted  its 
slow  reappearance  when  the  animals  were  given  their  usual  food.  Bate 
and  Westwood  (1869)  stated  that  the  color  of  Idotea  is  influenced  by 
the  food,  in  that  animals  which  eat  Fucus  are  dark  or  black,  while 
those  which  eat  green  algae  are  green.  This  statement,  however,  has 
been  denied  by  Mobius  (1873)  and  Matzdorff  (1883).  Beddard 
(1892)  cited  the  observation  of  Eisig  that  certain  marine  worms  be- 


186  CAROTINOIDS  AND  RELATED  PIGMENTS 

come  colored  by  the  lipochromes  of  the  sponge  upon  which  they  live 
as  a  parasite.  Dastre  (1899)  noticed  that  he  could  suppress  a 
chlorophyll-like  pigment  which  occurs  normally  in  the  liver  of  mol- 
luscs by  withholding  chlorophyll-containing  food,  and  that  the  pig- 
ment was  also  absent  from  the  liver  at  the  end  of  the  hibernation 
period.  Villard  (1903)  and  Przibram  (1906,  1907,  1909)  found  that 
leaf  lice  which  are  raised  on  etiolated  plants  in  the  dark  are  mostly 
pale  yellow.  Schneider  (1908)  mentions  that  he  found  that  the  crab- 
eating  perch,  Perca  fluviatilis,  takes  on  the  characteristic  red  color  of 
the  crab  in  various  places  on  its  body.  He  thought  that  the  pigment, 
which  he  speaks  of  as  crustaceorubin,  took  the  place  of  a  red  pigment 
which  normally  colors  the  fish,  but  it  seems  more  likely  that  the 
normal  red  pigment  of  the  fish  is  the  carotinoid  derived  from  its  food. 

A  still  more  striking  as  well  as  very  recent  instance  of  biological 
relationship  affecting  lipochromes  is  mentioned  by  Gerould  (1921)  in 
connection  with  a  blue-green  mutation  of  the  normally  grass-green 
caterpillar  Colias  {Eurynvus)  Philadice,  the  blue  mutant  lacking 
the  normal  lipochrome  in  its  hemolymph,  eye,  cuticle,  etc.,  and  the 
eggs  of  the  butterfly  from  the  blue  mutant  being  pure  white  instead 
of  the  usual  yellow.  The  biological  relationship  involving  the  lipo- 
chrome is  between  the  caterpillar  and  the  color  of  the  cocoons  spun 
by  the  parasite  Apanteles  flaviconchce  which  emerges  from  it.  These 
cocoons  are  normally  yellow  from  the  normal,  lipochrome  containing 
grass-green  caterpillar,  but  were  pure  white  from  the  blue-green 
mutant  which  lacked  lipochrome.  According  to  Gerould,  "Yellow 
blood  in  silkworms  is  closely  correlated  with  the  spinning  of  yellow 
silk,  white  blood  with  white  silk,  Ude  (1919),  however,  has  dis- 
covered a  strain  of  yellow  stock  that  spins  white  silk,  although  their 
silk  glands  are  yellow." 

All  of  the  above  citations  support  the  idea  of  a  relationship  between 
plant  and  animal  pigments  and  even  between  pigments  among  animals, 
which  is  more  than  a  mere  chemical  relationship  or  identity.  An 
especially  striking  argument  supporting  the  existence  of  a  general 
biological  relation  such  as  is  suggested  by  these  instances  is  furnished 
by  the  fact,  pointed  out  by  Beddard  (1892),  that  there  is  a  uniform 
absence  of  pigment  from  cave  animals  coincident  with  the  absence  of 
chlorophyll  from  cave  plants. 

The  number  of  scattered  observations  supporting  this  thesis  is  fairly 
gratifying.  Prior  to  the  work  of  Palmer  and  Eckles  (1914),  however, 
very  few  definite  experiments  were  carried  out  which  show  the  possi- 


BIOLOGICAL  RELATIONS  BETWEEN  CAROTINOIDS    187 

bility  of  transferring  carotinoid  pigment  from  plants  to  animals,  or 
which  were  designed  to  determine  whether  any  of  the  normal  pigments 
of  animals  are  merely  derived  from  the  food. 

The  earliest,  as  well  as  the  most  interesting  experiment  of  this 
kind,  so  far  as  carotinoids  are  concerned,  was  conducted  by  Sauermann 
(1880)  who  studied  the  effect  of  feeding  cayenne  pepper  to  birds.  He 
became  interested  in  the  problem  because  of  the  custom  in  vogue  at 
that  time  of  coloring  the  feathers  of  canary  birds  by  feeding  them  red 
pepper.  It  is  stated  in  the  paper  that  the  canary  bird  dealers  who 
practiced  this  artificial  coloring  mixed  the  cayenne  pepper  with  egg 
yolk  and  bread  and  fed  the  mixture  to  the  very  young  birds  or  to  old 
birds  during  the  molting  season,  thereby  coloring  the  new  feathers 
a  yellow  to  red  color.  When  Sauermann  tried  the  experiment  using 
the  red  pods  from  which  the  pepper  but  not  the  pigment  had  been 
extracted  with  60  per  cent  alcohol,  the  pepper  plants  had  scarcely  any 
effect  on  the  color  of  the  plumage.  The  same  result  followed  the  feed- 
ing of  the  crude  pigment  which  had  been  extracted  with  absolute 
alcohol,  but  when  this  extract  was  dissolved  in  sunflower  oil  the  re- 
sults reported  by  the  canary  bird  dealers  were  confirmed. 

Especially  interesting  were  Sauermann's  experiments  on  feeding  the 
pepper  pigment  to  fowls.  In  this  case  the  cayenne  pepper  itself  was 
fed  to  12  white  Italian  fowls,  8  weeks  old,  the  young  chickens  being 
fed  25  grams  of  the  pepper  night  and  morning  mixed  with  moistened 
bread  and  potatoes.  The  birds  received  corn  and  oats  in  addition.  It 
is  stated  that  the  feet  of  all  the  fowls  became  orange  and  that  the 
pepper  pigment  could  be  extracted  from  them  by  soaking  them  in 
alcohol  for  a  long  time  and  then  extracting  with  ether.  No  proof  is 
given  for  the  fact  that  the  pigment  extracted  in  this  manner  was  the 
pepper  pigment.  Only  two  of  the  12  fowls  showed  any  effects  of  the 
pepper  feeding  on  the  feathers.  The  pigment  began  to  appear  on  the 
breast  of  one  hen  in  about  10  days  and  the  other  in  about  3  weeks. 
The  first  hen  eventually  developed  a  red  breast  and  the  rest  of  the 
body  became  yellowish  red,  but  the  second  hen  only  developed  red 
feathers  on  the  breast,  the  rest  of  the  body  remaining  white.  Old  hens 
were  not  influenced  in  the  least  by  pepper-feeding,  even  during  the 
molting  season.  If  the  pigment  which  appeared  on  the  feathers  of 
the  two  young  birds  was  the  red  pepper  pigment,  it  is  difficult  to 
understand  why  none  of  the  other  young  birds  were  affected,  or  why 
the  old  hens  did  not  develop  some  tint  in  the  new  feathers  formed 
during  the  molt. 


188  CAROTINOIDS  AND  RELATED  PIGMENTS 

Somewhat  more  convincing  were  Sauermann's  results  on  feeding  the 
red  pepper  to  laying  hens.  By  feeding  a  hen  5  grams  of  the  pepper 
each  day  the  pigment  appeared  in  the  third  egg  laid  after  the  beginning 
of  the  pepper  feeding,  as  a  thin  band  of  color  at  the  periphery  of  the 
yolk.  By  the  time  the  sixth  egg  was  laid  the  yolk  was  entirely  pig- 
mented. Two  interesting  properties  were  noticed  in  connection  with 
the  yolks  colored  by  the  pepper  pigment:  (1)  it  was  impossible  to 
hard-boil  them,  (2)  ether  would  not  extract  all  the  color  from  the  dried 
yolks,  because  a  part  of  the  pigment  was  apparently  bound  tightly  to 
the  protein. 

These  experiments  have  a  bearing  on  the  biological  relationship  be- 
tween plant  and  animal  carotinoids  for  two  reasons.  They  not  only 
record  the  first  authentic  instance  in  which  a  plant  carotinoid  was 
transferred  to  an  animal  under  experimental  conditions,  but  are  also 
the  only  experiments  showing  the  possibility  of  lycopin  occurring  in 
the  animal  body.  It  was  shown  in  Chapter  II  that  the  evidence  indi- 
cates that  the  chief  pigment  in  the  ripe  fruit  of  the  pepper  plant, 
Capsicum  annum,  is  the  red  carotin  isomer,  lycopin.  Presumably  this 
was  the  chief  pigment  in  the  cayenne  pepper  which  Sauermann  fed  to 
his  hens,  and  which  appeared  in  the  egg  yolks  and  in  the  feathers  in 
two  of  the  birds.  The  evidence,  although  circumstantial,  is  strongly 
in  favor  of  this  deduction,  and  should  be  submitted  to  further  verifi- 
cation because  the  result  presents  the  apparent  anomaly  that  lycopin, 
the  isomer  of  carotin,  can  be  transferred  abundantly  to  the  egg  yolk 
of  the  hen  while  carotin  appears  in  the  yolk  only  in  traces  even  under 
the  most  favorable  conditions. 

The  next  experiment  was  that  of  Poulton  (1893),  carried  out  to 
verify  his  previous  (1885)  hypothesis  that  the  colors  of  caterpillars 
are  due  largely  to  plant  pigments  derived  from  the  food.  Newly 
hatched  larvae  were  placed  on  three  diets:  (1)  yellow  etiolated  leaves 
from  the  center  of  a  heart  of  cabbage,  (2)  white  mid-rib  of  cabbage 
containing  no  pigment,  and  (3)  deep  green  external  leaves.  All  were 
kept  in  the  dark.  The  larvae  raised  on  the  green  leaves  and  the 
etiolated  leaves  grew  normally,  those  on  the  etiolated  leaves  growing 
far  more  rapidly  than  those  on  the  green  leaves.  Both  of  these  sets  of 
caterpillars  developed  the  normal  green  and  brown  colors  of  the 
species.  The  caterpillars  on  the  colorless  cabbage  did  very  poorly  and 
only  one  was  raised  to  adult  size.  This  individual,  however,  remained 
colorless  throughout  the  experiment.  Some  of  the  group  of  caterpillars 
on  the  colorless  food  were  placed  on  the  etiolated  leaves  after  growing 


BIOLOGICAL  RELATIONS  BETWEEN  CAROTINOIDS    189 

to  a  certain  size,  but  these  did  not  develop  normal  color.  It  is  espe- 
cially interesting  that  the  larvae  on  the  etiolated  leaves,  as  well  as 
those  on  the  green  leaves,  should  have  developed  green  color.  Poulton 
thought  that  this  indicated  that  the  pigment  of  the  etiolated  leaf  is 
closely  related  chemically  to  chlorophyll,  but  since  the  pigment  of 
the  etiolated  leaf  is  now  generally  regarded  as  consisting  chiefly  of 
carotin,  the  explanation  of  the  greening  of  the  caterpillars  must  lie 
in  their  failure  to  inhibit  the  development  of  chlorophyll  from  its  pre- 
cursors in  the  etiolated  leaf.  Poulton's  experiments  have  been  accepted 
as  having  proved  that  caterpillars  derive  their  green  and  yellow  pig- 
ments from  their  food.  In  fact,  it  is  generally  held  at  the  present  time 
that  all  phytophagous  insects  derive  their  lipochromes  and  chlorophyll- 
like pigments  from  their  food,  and  that  the  former,  at  least,  are  passed 
on  from  the  larvae  to  the  adults. 

Gamble  (1910)  attempted  to  determine  whether  the  pigments  which 
develop  in  the  hypodermis  of  the  young  crustacean  Hippolyte  varians, 
is  derived  from  the  food.  The  newly-hatched  larvae  of  this  species  are 
colorless  with  the  exception  of  lines  of  red  pigment  on  the  hypodermis. 
When  the  adolescent  larvse  are  placed  among  green  or  red  seaweeds, 
the  entire  hypodermis  will  turn  green  or  red  in  48  hours.  Gamble 
placed  the  colorless  adolescent  Crustacea  in  the  inner  chamber  of 
double-walled  glass  vessels,  and  put  a  mass  of  green  or  red  or  brown 
algsD  in  the  outer  chamber  and  fed  the  Crustacea  various  foods,  such 
as  etiolated  Laminaria,  red  crab  meat,  colorless  crab  ovaries,  and  red 
crab  ovaries.  At  the  end  of  several  days  the  Crustacea  had  in  most 
cases  developed  a  color  similar  to  their  surroundings  rather  than  like 
that  of  their  food.  The  conclusion,  therefore,  seems  justified  that  the 
red,  green  and  brown  colors  in  Hippolyte  are  not  derived  from  the 
food.  No  observation  seems  to  have  been  made,  however,  on  the 
yellow  pigment  which  occurs  in  the  chromatophores  of  the  fully  de- 
veloped Crustacea.  Gamble  states  that  true  yellow  pigment  is  absent 
from  the  chromatophores  of  the  newly  hatched  larvae. 

In  addition  to  the  experiments  of  Sauermann,  Poulton  and  Gamble, 
there  remains  to  be  mentioned  that  of  Dombrowsky  (1904)  who  ob- 
served that  the  milk  of  a  goat  was  tinted  by  feeding  it  carrots,  and 
also  that  of  Moro  (1908)  who  noticed  a  tinting  of  the  skin  of  children 
fed  bountifully  on  carrot  soup.  The  actual  transference  of  carotin 
from  the  food  to  the  tissues  and  secretions  of  man  and  the  higher 
animals  is  at  least  indicated,  although  not  demonstrated,  in  these  cases. 

The  writer's  attention  was  attracted  to  a  possible  biological  rela- 


190  CAROTINOIDS  AND  RELATED  PIGMENTS 

tionship  between  plant  and  animal  carotinoids  in  1912  in  order  to 
explain  the  quantitative  variations  in  the  pigmentation  of  butter  fat 
due  to  changes  in  the  ration  of  the  cow.  A  study  of  the  chemical  and 
physical  properties  of  the  butter  fat  pigment  had  shown  it  to  be  iden- 
tical with  carotin,  regardless  of  the  extent  of  the  pigmentation  of  the 
butter  fat  from  which  the  pigment  was  isolated.  The  more  highly 
tinted  fats,  however,  showed  the  presence  of  small  amounts  of  xantho- 
phylls  associated  with  the  carotin  when  the  total  pigment  was  exam- 
ined by  means  of  the  phase  test  or  analyzed  by  means  of  a  Tswett 
chromatogram.  When  these  facts  were  viewed  in  the  light  of  the  dis- 
tribution and  amount  of  carotinoid  pigments  in  the  usual  dairy  cattle 
foods,  and  when  numerous  data  on  the  variations  in  the  color  of  butter 
fat  under  known  feeding  conditions  were  interpreted  with  these  facts  in 
mind  the  conclusion  was  inevitable  that  the  carotinoids  of  butter  fat 
are  derived  from  the  carotinoids  of  the  food.  This  conviction  was 
strengthened  further  by  a  study  of  the  character  of  the  pigment  of 
the  adipose  tissue,  skin  secretions  and  especially  the  blood  serum  of 
dairy  cattle  showing  that  the  pigment  in  each  case  is  chiefly  carotin, 
with  which  a  small  amount  of  xanthophyll  is  usually  associated.  The 
preliminary  statement  of  Willstatter  and  Escher  (1912),  published 
during  the  course  of  these  studies,  that  the  corpus  luteum  pigment  of 
the  cow  is  also  carotin  (an  observation  which  we  were  able  to  con- 
firm) ,  lent  additional  support  to  the  theory  of  a  biological  relationship 
between  the  lipochromes  in  cattle  and  the  carotinoids  of  their  ration. 
The  correctness  of  this  theory  was  shown  by  varying  the  content 
of  carotinoids  in  the  ration  of  the  cow  through  the  proper  selection 
of  foods  deficient  in  carotinoids  or  containing  an  abundance  of  these 
pigments  and  observing  the  quantitative  variations  in  the  amount 
of  pigment  in  the  blood  serum  and  butter  fat.  These  experiments  were 
supplemented  by  an  examination  of  the  character  of  the  pigment  in 
the  blood  and  butter.  In  addition  two  dairy  cows  of  the  Jersey  breed, 
whose  adipose  tissue  is  normally  highly  pigmented  with  carotin,  were 
fattened  on  rations  respectively  rich  and  poor  in  carotin  after  a  pre- 
liminary period  of  sixty  days  on  straw  alone.  Some  of  the  data 
secured  in  the  experiments  designed  to  show  the  biological  relation 
between  the  carotin  content  of  the  blood  serum  and  milk  fat  and  that 
of  the  ration  are  given  in  Table  14.  Table  15  shows  the  effect  on  the 
color  of  the  adipose  tissue  in  certain  parts  of  the  body  of  Jersey  cattle 
which  results  when  partially  starved  animals  are  fattened  on  rations 
deficient  or  rich  in  carotin. 


BIOLOGICAL  RELATIONS  BETWEEN  CAROTINOIDS    191 


Table  14. — The  Relation  between  Carotin-rich  and  Carotin-pook  Rations  and 
THE  Color  of  Milk  Fat  and  Blood  Serum 

Breed  oj  Butter  Fat   Blood  Serum 

Cow  Ration  Yellow  Bed    Yellow  Bed 

Carotin-poor  rations 
Ayrshire  Cottonseed  meal  and  cottonseed  hulls        1.3 '^      0.4        3.3'      0.5 

"  Cottonseed  hulls,  timothy  hay  and  white 

corn  1.2        0.4        2.6        1.1 

"  Cottonseed     meal,     cottonseed     hulls, 

timothy  hay  and  yellow  corn  2.0        0.5        4.9        1.2 

Holstein  Cottonseed  hulls,  corn  stover  and  cot- 

tonseed meal  8.5        1.4        6.0        0.7 

"  Cottonseed  hulls,  corn  stover  and  cot- 

tonseed meal  3.0        0.7        7.0        0.8 

Ayrshire  Cottonseed  hulls,  corn  stover  and  cot- 

tonseed meal         .  2.5        0.6      11.0        0.9 

Jersey  Cottonseed  hulls,  corn  stover  and  cot- 

tonseed meal  11.0        1.7      10.0        0.9 

"  Cottonseed  hulls,  com  stover  and  cot- 

tonseed meal  5.2        1.2      13.0        1.1 

"  Cottonseed  hulls,  corn  stover  and  cot- 

tonseed meal  4.7        1.5        7.5        0.7 

Carotin-rich  rations 
Ayrshire  Cottonseed     meal,     cottonseed     hulls, 

timothy  hay,  yellow  corn  and  carrots        24.0        1.3      54.0        1.8 
"  Mixed    grain,    green    alfalfa    hay    and 

fresh  pasture  grass  16.0        1.1      40.0        1.0 

Holstein  Mixed    grain,    green    alfalfa    hay    and 

fresh  pasture  grass  54.0        1.8      48.0        1.1 

"  Mixed    grain,    green    alfalfa    hay    and 

fresh  pasture  grass  22.0        1.2      41.0        1.0 

Jersey  Mixed    grain,    green    alfalfa    hay    and 

fresh  pasture  grass  64.0        2.0      45.0        1.1 

"  Mixed    grain,    green    alfalfa    hay    and 

fresh  pasture  grass  54.0        1.7      57.0        1.8 

"  Mixed    grain,    green    alfalfa    hay    and 

fresh  pasture  grass  47.0        1.6      45.0        1.0 

Table  15. — The  Relation  between  Carotin-poor  and  Carotin-rich  Rations  and 
THE  Color  of  the  Adipose  Tissue  of  Dairy   Cattle  Deposited  during  the 

Feeding  of  These  Rations  _,  ,     ,     ,    .  ,.  ^. 

Color     oj  Adipose  Tissue 
Carotin-rich  Bation       Carotin-poor  Bation 
Source  of  Adipose  Tissue  Yellow  Bed  Yellow         Bed 

Inside  of  rib  50.0  2.3  1.4  0.1 

Mesentery    47.0  2.1  3.6  0.5 

Thoracic  cavity   29.0  1.3  8.0  1.0 

Around  ovaries  and  uterus   49.0  2.3  2.5  0.3 

Attached  to  omasum  33.0  1.6  24.0  1.7 

In  pelvic  cavity   50.0  2.3  47.0  2.1 

Around  kidney   54.0  1.6  50.0  2.1 

Over  last  rib   47.0  2.3  50.0  2.1 

Over  outside  chuck   47.0  2.0  47.0  1.8 

2  The  color  of  the  butter  fat  was  determined  by  matching  a  1-inch  layer  of  rendered 
melted  fat  with  the  color  glasses  of  the  Loviboud  tintometer. 

»  The  color  of  the  blood  serum  was  determined  by  matching  the  extract  from  10  cc. 
of  serum  in  12.5  cc.  volume  and  1-inch  layer  with  the  color  glasses  of  the  Lovibond 
tintometer. 

*  The  color  readings  were  taken  on  a  1-inch  layer  of  rendered,  melted  fat,  using  the 
Lovibond  tintometer. 


192  CAROTINOIDS  AND  RELATED  PIGMENTS 

The  experiments  demonstrated  conclusively  that  the  carotin  content 
of  the  cow's  tissues  as  well  as  that  secreted  in  the  milk  fat  is  deter- 
mined by  the  carotin  content  of  the  ration.  One  of  the  interesting 
features  of  the  experiments  was  the  demonstration  that  this  relation 
is  independent  of  the  breed  of  the  cow;  just  as  striking  changes  in 
the  pigmentation  of  the  milk  fat,  blood  serum  and  adipose  tissue  were 
brought  about  in  the  highly  colored  breeds  as  in  those  which  are  not 
usually  so  highly  pigmented.  So  far  as  the  blood  serum  is  concerned 
the  breed  appears  to  have  no  bearing  on  the  maximum  carotin  con- 
tent, as  the  data  in  Table  14  show.  This  fact,  together  with  a  certain 
lack  of  parallelism  between  the  changes  in  the  carotin  content  of  the 
blood  and  corresponding  changes  in  the  carotin  content  of  the  milk 
fat  indicates  that  the  breed  differences  involving  the  color  of  the  milk 
fat  and  adipose  tissue  are  determined  at  the  site  of  the  synthesis  of 
the  milk  fat  and  adipose  tissue.  It  is  not  at  all  improbable  that  the 
carotin- albumin  complex  which  carries  the  carotin  in  the  blood  serum 
plays  a  prominent  part  in  controlling  these  differences. 

A  surprising  feature  of  the  experiments  was  the  failure  of  a 
xanthophyll-rich  cattle  food,  such  as  yellow  maize,  to  exert  any  appre- 
ciable influence  on  the  color  of  butter  fat.  This  is  brought  out  clearly 
in  Table  14.  In  the  experiment  reported  in  that  table  the  ration  con- 
tained 6  pounds  of  yellow  maize  daily.  In  other  experiments  re- 
ported by  Palmer  and  Eckles  (1914a)  as  much  as  12  pounds  of  yellow 
maize  was  fed  without  effect.  These  results  are  contrary  to  popular 
opinion  (compare  Newbigin,  quoted  above),  but  are  unquestionably 
explained  by  the  fact  that  carotin  is  only  a  minor  fraction  of  the 
pigment  of  yellow  maize,  the  major  pigment  being  xanthophyll,  which 
appears  to  play  very  little  part  in  coloring  the  tissues  or  fluids  of 
dairy  cattle. 

A  word  should  perhaps  be  said  regarding  the  experiment  whose 
results  are  summarized  in  Table  15.  It  is  obvious  on  inspecting  these 
data  that  only  certain  parts  of  the  body  were  affected.  This  is  ex- 
plained by  the  fact  that  the  preliminary  starvation  period  of  the 
animals  failed  to  remove  appreciable  amounts  of  fats  from  the  out- 
side of  the  body  or  from  around  some  of  the  vital  organs.  It  seems 
evident  that  the  mesentery  and  related  fats  were  drawn  upon  chiefly 
during  the  period  of  partial  starvation  because  it  was  the  fat  deposited 
in  these  parts  during  the  fattening  period  that  was  affected  by  the 
carotinoid-deficient  ration. 

Palmer   (1915)    carried  out  similar  experiments  with  fowls.     The 


BIOLOGICAL  RELATIONS  BETWEEN  CAROTINOIDS    193 

results  not  only  confirmed  the  findings  of  Willstatter  and  Escher 
(1912)  as  to  the  xanthophyll  character  of  the  major  pigment  of  egg 
yolk,  but  demonstrated  as  well  that  the  same  pigment  is  found  in  the 
blood  serum  and  adipose  tissue.  The  results  of  the  earlier  studies  on 
the  origin  of  the  carotin  in  cattle  naturally  suggested  that  the  xantho- 
phyll in  the  tissues  of  the  fowl  and  in  the  yolks  of  its  egg  is  similarly 
derived  from  the  xanthophyll  of  the  food.  This  was  demonstrated  to 
be  the  case  in  carefully  controlled  feeding  experiments  in  which  a 
xanthophyll-rich  ration  (containing  an  abundance  of  yellow  maize), 
a  carotin-rich  ration  (containing  an  abundance  of  carrots)  and  a 
carotinoid-poor  ration  were  fed  to  laying  hens.  The  yolks  of  the 
eggs  increased  materially  in  color  on  the  xanthophyll-rich  ration,  and 
the  blood  serum  was  also  rich  in  pigment,  but  there  was  a  marked 
decline  in  the  color  of  the  egg  yolks  from  the  hens  on  the  carotin- 
rich  and  carotinoid-poor  rations  which  was  practically  parallel  and 
which  was  accompanied  by  almost  carotinoid-free  blood  serum.  The 
experiments  showed  very  clearly,  however,  that  there  is  not  an  abso- 
lute exclusion  of  carotin  by  the  hen;  the  egg  yolks,  adipose  tissue  and 
blood  serum  always  contained  a  small  proportion  of  the  total  pigment 
in  the  form  of  carotin,  which  was  clearly  somewhat  greater  in  the  yolks 
of  the  eggs  from  the  carrot-fed  hens. 

These  experiments  on  the  biological  relation  of  the  carotinoids  of 
fowls  to  the  carotinoids  of  the  food  found  complete  confirmation  in 
the  experiments  of  Palmer  and  Kempster  (1919  a,  b,  c)  in  which  a 
flock  of  White  Leghorn  fowls  was  raised  to  maturity  from  the  time  of 
hatching  on  rations  so  devoid  of  carotinoids  that  the  mature  birds 
showed  only  the  merest  traces  of  pigment  in  adipose  tissue  and  no 
demonstrable  amounts  in  the  blood  serum  or  skin  and  none  in  the 
yolks  of  the  eggs  laid  by  the  mature  hens.  Xanthophyll-rich  feeds 
brought  about  a  rapid  coloration  in  all  parts  of  the  body  and  in  the 
egg  yolks  (except  in  the  case  of  laying  hens  when  the  egg  yolks  only 
were  colored)  while  carotin,  fed  in  the  form  of  highly  colored 
(colostrum)  butter  fat  had  practically  no  effect  on  the  color  of  the 
bird's  tissues. 

It  was  found  in  connection  with  the  writer's  (1914e)  milk  fat 
studies  that  the  pigment  of  human  milk  fat  consists  of  both  carotin 
and  xanthophyll.  By  analogy  with  the  cattle  experiments  it  was  con- 
cluded that  the  lipochromes  in  the  human  body  are  likewise  derived 
from  the  carotinoids  of  the  diet.  Hess  and  Myers  (1919)  later  demon- 
strated this  to  be  the  case  in  experiments  in  which  it  was  shown  that 


194  CAROTINOIDS  AND  RELATED  PIGMENTS 

the  skin  of  infants  can  be  colored  with  carotinoids  by  feeding  diets 
rich  in  carotin  or  xanthophyll,  and  that  this  was  accompanied  by  an 
increased  lipochrome  content  of  the  blood  serum.  The  latter  was 
identified  as  carotin  in  the  case  of  carrot  feeding.  Hess  and  Myers 
state  that  the  skin  pigmentation  resulted  in  infants  "when  the  dietary 
included  two  oranges  a  day,  or  the  yolk  of  one  egg  in  the  milk  formula 
for  a  period  of  two  months,  or  two  ounces  of  spinach  daily  for  a 
month,"  as  well  as  when  the  equivalent  of  two  tablespoonfuls  of  fresh 
carrots  was  fed  each  day  for  a  period  of  four  to  six  weeks.  An  in- 
crease in  the  carotin  content  of  the  blood  serum  was  also  noted  fol- 
lowing a  subcutaneous  injection  of  carotin  (from  carrots)  dissolved  in 
olive  oil,  and  the  pigment  also  appeared  in  the  urine.  The  latter  phe- 
nomenon was  noted  also  when  a  concentrated  carotin  solution  in  olive 
oil  was  given  to  an  infant  by  mouth. 

The  general  fact  that  man  and  the  higher  mammals  and  the  fowls 
deriA^e  the  chromolipoids  of  their  tissues  and  secretions  from  the  caro- 
tinoids of  their  food  is  now  well  established.  Van  den  Bergh,^  Muller 
and  Broekmeyer  (1920),  especially,  have  contributed  much  valuable 
data  on  the  variations  in  the  carotinoids  in  the  human  body  under 
normal  and  diseased  conditions,  as  well  as  contributing  observations 
on  the  character  and  extent  of  the  carotinoid  pigmentation  in  various 
species  of  animals.  The  numerous  reports  of  the  skin  colorations  of 
diabetics,  known  as  Xanthosis,  Carotinemia,  Lipochromemia,  etc., 
which  were  cited  briefly  in  Chapter  IV,  also  support  and  confirm  the 
biological  relationship  between  plant  and  animal  carotinoids,  at  least 
for  the  higher  animals.  Since  this  has  already  been  demonstrated 
to  be  the  case  for  the  phytophagous  insects  there  seems  to  be  no  valid 
reason  for  rejecting  the  general  thesis  that  all  animal  lipochromes 
are  derived  from  the  carotinoids  of  the  food.  This  conclusion  must 
be  accepted  for  the  herbivorous  animals  of  all  species,  both  vertebrates 
and  invertebrates,  in  which  lipochromes  are  found.  When  one  con- 
siders that  those  animals  which  prey  solely  on  lower  forms  of  animal 
life  usually,  if  not  always,  select  their  food  among  species  which  are 
herbivorous,  then  the  possibility  becomes -practicable,  if  not  demon- 

•  In  this  paper  van  den  Bergh  lays  claim  to  an  unpublished  study  carried  out  by  him 
in  1913  in  which  he  showed  that  the  lipochrome  content  of  the  blood  serum  of  men, 
fowls  and  cattle,  as  well  as  the  milk  of  the  latter  varies  with  the  lipochrome  content 
of  their  food.  In  his  splendid  paper  with  Snapper  (see  van  den  Bergh  and  Snapper 
[1913])  on  the  lipochrome  of  the  blood  serum  of  man,  horses  and  cattle,  he  shows 
unmistakably,  however,  that  he  regarded  the  pigment  as  originating  in  the  body, 
although  he  does  raise  the  question  as  to  the  origin  of  the  high  pigmentation  which  he 
observed  in  the  case  of  diabetics. 


BIOLOGICAL  RELATIONS  BETWEEN  CAROTINOIDS    195 

strated,  that  there  is  a  universal  dependence  on  the  diet  for  the  lipo- 
chrome  carotinoids  of  animal  tissues.  One  cannot,  in  fact,  account  in 
any  other  way  for  the  brilliant  carotinoid  colorations  among  certain 
birds,  fish  and  other  lower  vertebrates. 

It  must  not  be  forgotten,  however,  that  not  all  animal  lipochromes 
in  the  broad  sense  have  been  definitely  identified  as  carotinoids. 
There  is  at  least  one  (possibly  more)  red  lipochrome  widely  distributed 
among  animals,  as  shown  in  Chapter  IV,  which  does  not  seem  to  have 
an  analogue  among  the  plant  carotinoids,  although  it  seems  to  be 
closely  related  to  them.  Such  a  pigment  occurs  in  the  feathers  of 
certain  birds,  in  the  salmon  muscle,  in  the  hypoderm  of  Crustacea  and 
elsewhere.  Is  it  a  plant  carotinoid  which  has  not  yet  been  identified, 
or  is  it  a  modified  plant  carotinoid?  Either  of  these  possibilities  is 
more  rational  than  the  possibility  that  it  is  actually  synthesized  by 
the  animals  in  which  it  occurs.  The  red  pigment  on  the  legs  of  the 
pigeons  is  probably  such  a  pigment.  It  is  absent  from  the  legs  of  the 
young  pigeons.  In  the  color  of  its  solutions  this  pigment  strongly 
resembles  lycopin.  The  writer  observed  recently  that  the  pigment 
is  strongly  epiphasic  between  petroleum  ether  and  90  per  cent  methyl 
alcohol,  but  its  solutions  show  no  clear  absorption  spectra.  These 
tests  indicate  a  modified  carotin,  and  yet  there  is  little  if  any  carotin 
in  the  blood  serum  of  the  pigeon,  the  great  bulk  of  the  pigment  being 
xanthophyll  as  in  the  case  of  the  fowl.  The  problem  of  modified  caro- 
tinoids in  animal  tissues  is  therefore  an  important  phase  of  the  general 
hypothesis  which  must  not  be  overlooked. 

There  is  still  another  phase  of  animal  pigmentation  from  the  caro- 
tinoid standpoint  which  deserves  consideration,  namely,  that  caroti- 
noid pigmentation  is  not  universal,  even  among  herbivorous  animals. 
The  writer  (1916)  first  called  attention  to  some  variations  of  this 
kind  among  mammals,  showing  that  practically  no  carotinoids  occur 
in  sheep  and  goats  and  none  in  swine.  Palmer  and  Kennedy  (1921) 
showed  that  there  are  none  of  these  pigments  in  the  albino  rat.  Ro- 
dents in  general,  however,  do  not  lack  carotinoids,  for  one  finds  small 
amounts  in  the  guinea  pig,  as  shown  by  van  den  Bergh,  Muller  and 
Broekmeyer  (1920).  The  rabbit  is  practically,  if  not  entirely  devoid 
of  carotinoids,  although  entirely  herbivorous.  The  same  seems  to  be 
true  of  the  dog,  which  is  carnivorous,  at  least  by  preference.  Cats, 
however,  contain  traces,  as  shown  by  van  den  Bergh  and  associates 
(1920).  The  general  observation  that  some  animals  lack  lipochromes 
is  not  new,  for  Miss  Newbigin  used  it  as  an  argument  against  the 


196  CAROTIN OWS  AND  RELATED  PIGMENTS 

hypothesis  that  the  lipochromes  are  derived  pigments,  as  shown  in 
the  quotation  given  above  from  her  (1898)  paper  on  salmon  pigments. 
The  fact  is  none  the  less  puzzling,  however,  and  offers  a  very  attrac- 
tive problem  for  research.  There  is  evidently  a  physiological  factor 
involved  which  is  characteristic  of  the  species  and  thus  transmitted. 
Is  it  an  enzyme,  possibly  an  oxidase,  in  the  digestive  tract,  blood 
stream  or  a  vital  organ,  as  Gerould  (1921)  believes  to  be  the  case 
for  the  carotinoid-free  mutant  which  he  has  discovered  from  a  nor- 
mally carotinoid-containing  caterpillar?  If  the  carotinoid-free  species 
of  animals  possess  a  more  highly  developed  means  of  oxidizing  the 
carotinoids  introduced  in  their  food  it  should  be  possible  to  determine 
this  fact.  If  the  site  of  this  destruction  is  in  the  digestive  tract  the 
faces  of  these  animals  should  be  devoid  of  the  pigments  when  the  ani- 
mals are  on  carotinoid-rich  diets.  These  ideas  merely  give  a  hint  of 
the  modes  of  attacking  this  problem  which  suggest  themselves  to  the 
physiological  chemist. 

An  even  more  fascinating  problem  is  offered  by  the  fact  that  the 
cow  and  the  horse  resorb  the  carotin  of  their  rations  to  the  relative 
exclusion  of  the  xanthophylls  although  the  latter  are  the  more 
abundant  in  their  food,  whereas  the  fowl  resorbs  xanthophylls  to  the 
relative  exclusion  of  carotin.  The  failure  of  cows  to  respond  to  the 
feeding  of  xanthophyll  and  the  inability  of  the  hen  to  transmit  appre- 
ciable amounts  of  carotin  into  the  egg  yolk  shows  that  these  results 
are  not  to  be  explained  on  the  grounds  that  these  two  species  of  ani- 
mals have  the  power  to  convert  one  carotinoid  into  the  other.  Palmer 
and  Eckles  (1914d)  published  the  results  of  an  attempt  to  determine 
whether  there  is  a  greater  destruction  of  xanthophyll  than  carotin 
along  the  digestive  tract  of  the  cow  and  whether  there  is  any  differ- 
ence between  the  action  of  the  natural  and  artificial  digestive  fluids  on 
these  two  classes  of  carotinoids.  In  general,  the  results  throw  very 
little  light  on  the  fate  of  the  carotinoids  during  digestion  although 
carotin  appeared  to  show  a  greater  stability;  the  most  significant 
result  secured  was  that  bile  dissolves  amorphous  xanthophyll  deposits 
very  readily,  while  carotin  residues  are  taken  up  very  slowly.  This 
may  indicate  that  the  xanthophylls  are  transported  to  the  liver  and 
there  become  oxidized  while  carotin,  which  forms  a  complex  with  a 
blood  protein,  escapes  this  fate.  Confirmation  of  the  low  solubility  of 
carotin  in  bile  is  seen  in  the  finding  of  Fischer  and  Rose  (1913)  that 
the  gall  stones  of  cows  contain  crystallizable  carotin. 

No  similar  studies  have  ever  been  undertaken  with  fowls.     The 


BIOLOGICAL  RELATIONS  BETWEEN  CAROTINOIDS     197 

determination  of  the  relative  solubility  of  their  bile  on  carotin  and 
xanthophylls  might  lead  to  suggestive  results.  No  fact  has  as  yet 
come  to  light,  however,  which  offers  any  reasonable  basis  upon  which 
one  can  construct  an  explanation  of  the  rather  astonishing  divergence 
between  this  species  and  the  mammals  with  respect  to  the  class  of 
carotinoids  which  predominate  in  the  chromolipoids  which  color  the 
body  tissues. 

Summary 

The  demonstration  of  the  possibility  of  a  general  biological  rela- 
tionship between  animal  chromolipoids  and  plant  carotinoids  is  a 
recent  achievement.  Such  a  relationship  was  suggested  by  earlier, 
workers  in  isolated  cases,  but  even  the  chemical  identification  of  cer- 
tain of  the  animal  lipochromes  with  plant  carotinoids  did  not  suggest 
to  Willstatter  and  his  pupil  Escher  their  possible  origin  from  plant 
pigments. 

Of  the  earlier  investigators  Krukenberg  and  Zopf  saw  no  evidence 
of  such  a  biological  relationship.  Miss  Newbigin  concluded  that  such 
a  relation  existed  in  specific  cases  but  was  not  general.  Poulton,  how- 
ever, decided  that  for  caterpillars  the  yellow  pigment  is  derived  from 
"xanthophyll"  and  the  green  from  chlorophyll. 

In  addition  to  Poulton's  work,  the  earlier  experiments  demonstrating 
that  plant  carotinoids  can  be  transferred  to  animal  tissues  included 
Sauermann's  (1889)  coloring  of  the  feathers  of  canary  birds  and  fowls, 
and  the  egg  yolks  of  the  latter,  with  red  pepper  pigment.  The  experi- 
ments should  be  repeated,  however,  because  the  results  present  the 
apparent  anomaly  that  lycopin  (which  is  apparently  the  red  pepper 
pigment),  the  isomer  of  carotin,  is  assimilated  by  the  fowl  while 
carotin  is  absorbed  only  in  traces  under  the  most  favorable  conditions. 

Since  these  earlier  studies  Palmer  (1914,  1915,  1919)  has  demon- 
strated that  the  carotin  of  the  butter  fat,  adipose  tissue,  blood  serum, 
skin  secretions,  etc.,  of  cattle  is  biologically  derived  from  the  food; 
that  a  similar  relationship  exists  between  the  xanthophyll  of  egg  yolk 
and  fowl  tissues  and  plant  xanthophyll;  that  while  there  is  not  an 
absolute  exclusion  of  xanthophyll  by  the  cow  or  carotin  by  the  hen, 
the  occurrence  of  a  predominating  type  of  carotinoid  in  each  of  the 
two  species  is  not  due  to  the  power  of  the  animals  to  convert  one 
type  of  pigment  into  the  other;  and  that  the  pigments  of  human  milk 
fat  (and  presumably  of  human  tissues  in  general)  may  contain  either 
carotin  or  xanthophylls  or  both. 


198  CAROTINOIDS  AND  RELATED  PIGMENTS 

These  results  have  been  confirmed  by  subsequent  investigations  by 
others  and  support  the  thesis  that  all  animal  chromolipoids  are  derived 
from  the  carotinoids  of  the  food,  and,  either  unchanged  or  slightly 
modified,  are  the  cause  of  the  yellow  to  red  chromolipoid  colors  of  all 
species  of  animals. 

Carotinoid  pigmentation  of  animal  tissues  is  not,  however,  universal, 
even  among  animals  whose  diet  is  normally  rich  in  these  pigments,  as 
shown  by  Palmer  (1916).  This  fact  offers  a  very  attractive  prob- 
lem for  research.    Several  methods  are  suggested  for  attacking  it. 


Chapter  VIII 
Methods  of  Isolation  of  Carotinoicls 

The  isolation  of  the  various  carotinoid  pigments  is  attended  with 
certain  difficulties,  which  are  chiefly  mechanical,  even  if  one  desires 
to  secure  only  a  few  grams  of  pure  crystals.  The  pigments  are  all 
quite  intense^  so  that  one  is  readily  deceived  by  the  color  as  to  the 
actual  amount  of  pigment  which  is  present.  This  fact  makes  it  neces- 
sary to  carry  out  the  operations  involved  on  a  rather  generous  scale, 
in  order  that  the  yields  may  justify  the  effort.  The  difficulties  from 
a  chemical  point  of  view  are  due  primarily  to  the  great  ease  of  oxida- 
tion of  the  pigments  and  secondarily  to  the  presence  of  colorless  lipoid 
impurities  which  unavoidably  contaminate  the  crude  products  because 
of  the  necessity  of  using  the  lipoid  solvents  for  the  extraction  process. 
The  great  ease  of  oxidation  of  the  carotinoids  requires  the  employ- 
ment of  vacuum  in  carrying  out  all  concentrations  and  the  use  of 
inert  gases,  if  possible,  during  crystallization  processes.  The  removal 
of  "lipoid  impurities  naturally  depends  somewhat  on  the  nature  of 
the  contaminating  substances.  Where  relatively  large  amounts  of 
glycerides  are  involved  it  is  necessary  to  resort  to  saponification  and 
subsequent  extraction  of  the  unsaponified  pigment.  As  far  as  carotin 
is  concerned,  or  its  isomer  lycopin,  this  can  be  done  without  injury  to 
the  pigment.  There  may  be  some  question  whether  or  not  certain  of 
the  xanthophylls  are  altered  slightly  by  this  process.  For  the  caro- 
tinoid fucoxanthin,  however,  saponification  should  certainly  be  avoided 
as  it  is  known  to  form  a  compound  with  alkalis  under  certain  con- 
ditions. The  sterols  are  removed  by  washing  the  crystals  with  cold 
solvents,  depending  upon  the  carotinoid  involved.  For  carotin,  cold 
alcohol  (absolute  or  98  per  cent)  is  best,  and  for  xanthophyll  cold 
petroleum  ether  (b.  p.  40-60°  C).  Recrystallization  must  of  neces- 
sity be  resorted  to  for  the  final  purifications.  The  details  of  the 
operations  are  mentioned  below. 

1  According  to  Arnaud  (1887)  carotin  is  still  visible  in  carbon  disulfide  in  1  part  per 
million  of  solvent. 

199 


200  CAROTINOIDS  AND  RELATED  PIGMENTS 

Isolation  of  Carotin 

Carrots.  The  carrot  root  naturally  suggests  itself  as  the  most 
available  source  of  the  pigment  carotin.  Several  methods  have  been 
proposed  by  various  investigators,  each  of  which  may  be  indicated 
briefly. 

The  method  of  Arnaud  (1886)  was  to  submit  the  fresh  grated  car- 
rots to  heavy  pressure  and  add  an  excess  of  neutral  lead  acetate  to 
the  juice.  The  precipitate  was  filtered  off,  dried  in  vacuum  and  added 
to  the  pressed  carrot  pulp,  which  had  also  been  dried.  The  combined 
material  was  then  washed  with  carbon  disulfide  at  a  low  temperature. 
Crude  carotin  crystallized  out  of  this  extract  on  concentrating  it  to  a 
low  volume  and  allowing  it  to  stand,  if  sufficient  material  had  been 
used.  Arnaud  obtained  three  grams  from  100  kgs.  of  carrots  by  this 
method.  He  states  that  most  of  the  impurities  could  be  washed  away 
from  the  crude  crystalline  material  by  cold  petroleum  ether.^  Final 
purification  was  secured  in  Arnaud's  work  by  dissolving  the  crystals 
in  the  least  possible  amount  of  carbon  disulfide  and  then  adding  a 
large  excess  of  absolute  alcohol,  in  which  carotin  is  practically  in- 
soluble. This  was  followed  by  a  spontaneous  crystallization  from 
cold  pertoleum  ether,  a  final  washing  with  cold  absolute  alcohol,  and 
drying  in  vacuum. 

Kohl's  (1902e)  method  for  isolating  carotin  from  carrots  offers 
certain  advantages  over  that  of  Arnaud,  particularly  because  smaller 
quantities  of  extraction  solvent  are  required.  The  carrots  are  sliced 
and  then  boiled  and  pressed.  The  writer  has  noticed  that  there  is 
practically  no  loss  of  pigment  in  either  the  water  in  which  the  carrots 
are  boiled  or  in  the  press  juice,  inasmuch  as  the  heat  coagulation  of 
the  proteins  seems  to  fix  the  carotin  in  the  tissues.  Kohl  washed  the 
first  press  cake  with  cold  alcohol,  pressed  it  again,  ground  it  and 
allowed  it  to  dry  in  the  air.  A  bright  orange-red  powder  resulted 
if  well  colored  carrots  were  chosen.  Kohl  extracted  this  powder  with 
ether  in  an  extractor  of  the  continuous  type  until  all  the  pigment  was 
extracted.  The  ether  was  removed  by  evaporation,  and  saponification 
carried  out  in  the  extraction  flask  by  boiling  for  an  hour  with  alcoholic 
potash.  The  evaporation  of  the  alcohol  was  carried  out  in  the  same 
flask  in  a  current  of  CO2,  and  the  dried  soap  extracted  with  chloro- 

"  It  is  almost  necessary  to  use  a  fat  solvent  in  this  case  because  of  tbe  high  content 
of  oil  in  the  carrot  root.  It  is  to  be  expected,  also,  that  some  pigment  will  be  lost  in 
carrying  out  the  operation. 


M^tHObS  OF  ISOLATION  OF  CAROTINOIDS         20i 

form.  The  carotin  was  precipitated  from  the  chloroform  by  an  excess 
of  absolute  alcohol  and  purified  by  recrystallization  from  petroleum 
ether.  Kohl  found  that  a  very  good  yield  of  crystals  could  be  secured 
by  omitting  the  saponification  process  and  merely  allowing  the  con- 
centrated ether  extract  to  evaporate  spontaneously.  The  crystals 
secured  in  this  manner  could  be  purified  further  by  washing  with  cold 
ether  followed  by  cold  absolute  alcohol. 

On  forming  a  concentrated  chloroform  solution  of  this  residue  and 
adding  three  volumes  of  absolute  alcohol,  the  impurities  which  pre- 
cipitated immediately  could  be  removed  by  a  quick  filtration.  On 
allowing  the  solution  to  stand  for  about  24  hours,  pure  carotin  crystal- 
lized out.  Kohl  does  not  tell  what  yields  he  secured  by  this  method, 
but  he  assures  us  that  the  product  was  of  a  high  degree  of  purity. 

A  method  somewhat  similar  to  that  of  Kohl  was  followed  by  Euler 
and  Nordenson  (1908).  Fresh  carrots  in  25  kg.  lots  were  boiled  in 
water  for  several  hours  and  then  pressed.  The  press  cake  was  ground 
with  sand  and  dried  in  thin  layers  at  50°  C,  which  took  about  a  day. 
The  dried  residue  was  extracted  twice  with  carbon  disulfide  at  20°  C, 
presumably  by  agitating  with  the  solvent.  The  volume  of  solvent 
used  was  not  stated.  The  carbon  disulfide  was  pressed  out  of  the 
dried  carrot  pulp  and  the  solvent  distilled  off  of  the  filtrate,  at  the 
last  with  the  addition  of  much  ether.  The  carrot  pulp  was  now  treated 
with  8  liters  of  alcohol  for  several  hours,  which  became  deep  red  with 
extracted  pigment.  By  diluting  with  much  water  and  shaking  with 
ether  the  pigment  was  transferred  to  the  latter  solvent.  The  two 
ether  solutions  of  pigment  were  treated  alike.  They  were  first  evapo- 
rated to  dryness  and  then  taken  up  in  a  little  petroleum  ether  and 
three  volumes  of  alcohol  added.  The  precipitated  phosphatides  were 
filtered  off  and  the  filtrates  evaporated  to  dryness.  The  combined 
yields  of  crude  pigment  amounted  to  26  grams. 

The  most  satisfactory  yields  of  pure  pigment  were  reported  by 
Escher  (1909)  who  obtained  125  grams  from  472  kgs.  of  dried  (5,000 
kgs.  fresh)  carrots.  The  complete  details  of  the  method  used  have 
not  been  accessible  to  the  writer.  In  general,  however,  Escher  dried 
the  carrots  without  previous  cooking,  using  a  low  heat.  The  dried 
pulp  was  ground  to  a  powder  and  the  pigment  completely  extracted 
by  petroleum  ether  in  a  continuous  extractor.  This  extract  was  con- 
centrated to  a  low  volume  under  diminished  pressure  at  40°  C.  On 
standing,  the  carotin  crystallized  out  together  with  a  large  amount  of 
colorless  impurities.    Purification  was  carried  out  by  fractional  pre- 


202  CAROTINOIDS  AND  RELATED  PIGMENTS 

cipitation  from  carbon  disulfide  solution  with  absolute  alcohol.  In 
this  process  the  colorless  impurities  precipitate  first  and  then  the  pure 
carotin.  By  repeating  the  fractional  precipitation  pure  carotin  was 
finally  obtained. 

Green  leaves.  Arnaud  (1885)  was  one  of  the  first  to  show  that 
crystals  of  carotin  may  be  secured  by  a  gentle  and  rapid  petroleum 
ether  extraction  of  vacuum  dried,  powdered  leaves,  e.g.,  spinach,  fol- 
lowed by  spontaneous  evaporation  of  the  concentrated  extract. 
Arnaud  found  that  the  waxy  substances  could  be  washed  away  with 
a  little  cold  ether  and  the  pigment  recrystallized  from  petroleum 
ether.  The  interesting  feature  of  this  method  is  the  fact  that  quick 
extraction  of  the  perfectly  dry  powdered  leaves  removes  practically 
no  green  pigment,  and  also  no  appreciable  amount  of  xanthophylls. 

Willstatter  and  Mieg  (1907)  applied  the  method  of  Arnaud  to  the 
leaves  of  the  stinging  nettle,  Urtica  dioica,  in  order  to  isolate  carotin 
on  a  large  scale.  The  nettle  leaves  are  not  so  good  a  source  of  pig- 
ment, however,  as  spinach,  according  to  these  investigators,  but  their 
low  yield  of  carotin  may  have  been  due  to  the  fact  that  the  leaves 
were  harvested  in  July  when  their  carotin  content,  according  to  Arnaud 
(1889),  is  quite  low.  The  details  of  the  operation  should  be  useful 
for  the  isolation  of  carotin  from  any  green,  leafy  material  containing 
a  relatively  large  quantity  of  the  pigment.  One  hundred  kgs.  of  pow- 
dered nettle  leaves  (moisture  7.7  per  cent)  were  allowed  to  stand  in  con- 
tact with  120  liters  of  cold  petroleum  ether  (b.  p.  40-70°  C.)  in  glass 
flasks  for  two  days.  The  petroleum  ether  was  filtered  off  on  a  Biichner 
funnel  and  the  residue  on  the  filter  washed  with  60  liters  of  petroleum 
ether.  It  is  stated  that  no  xanthophyll  was  present  in  the  greenish 
yellow  extract.  The  small  amount  of  chlorophyll  present  wag'  removed 
first.  This  was  done  by  shaking  the  extract  gently  with  a  little  con- 
centrated alcoholic  potash,  being  careful  to  avoid  an  emulsion.  The 
alkali  was  removed  by  washing  with  water,  but  here  again  care  had 
to  be  taken  to  shake  the  mixtures  very  gently  because  the  petroleum 
ether  solution  still  contained  considerable  fat-like  material.  In  the 
writer's  experience  these  processes  of  removing  the  chlorophyll  and 
washing  out  the  alkali  are  likely  to  be  somewhat  tedious.  When  they 
are  completed  one  can  proceed  to  the  evaporation  of  the  extracts, 
which  must  be  carried  out  in  vacuum.  In  Willstatter  and  Mieg's  ex- 
periments the  200  liters  of  petroleum  ether  were  evaporated  to  about 
three  liters  before  setting  aside  for  the  carotin  to  crystallize  out.    It 


METHODS  OF  ISOLATION  OF  CAROTINOIDS         203 

is  stated  that  when  the  petroleum  ether  had  evaporated  the  carotin 
was  found  as  glistening  crystals  embedded  in  a  dark,  waxy  mass. 

The  removal  of  the  waxy  impurities  and  the  final  purification  are 
carried  out  as  follows.  The  mixture  of  wax  and  pigment  is  carefully 
shaken  with  three  liters  of  the  lowest  boiling  petroleum  ether  (pre- 
sumably that  boiling  under  50°  C.)  which  removes  the  bulk  of  the 
wax.  The  solution  is  filtered,  leaving  the  carotin  on  the  filter  but 
still  somewhat  contaminated  with  phytosterol  .and  other  colorless  sub- 
stances. According  to  Willstatter  and  Mieg  the  carotin  lost  in  the 
filtrate  can  be  recovered  through  precipitation  by  alcohol,  but  the 
details  of  this  recovery  are  not  given.  The  final  purification  is  car- 
ried out  as  in  the  case  of  the  isolation  of  carotin  from  carrots,  namely, 
by  dissolving  in  a  small  amount  of  carbon  disulfide,  in  which  a  part 
of  the  colorless  substances  do  not  readily  dissolve,  and  then  by  adding 
absolute  alcohol  cautiously  to  secure  the  fractional  precipitation  of 
the  other  impurities  from  the  carotin.  The  colorless  substances  come 
down  first  and  can  be  quickly  filtered  off.  Then  the  carotin  precipi- 
tates as  sparkling  crystals.  At  this  point  the  yield  was  a  little  over  3 
grams  of  crystals  in  Willstatter.  and  Mieg's  work.  This  is  mentioned 
because  it  gives  a  good  idea  of  the  scale  on  which  it  is  apparently 
necessary  to  operate  in  order  to  secure  even  small  quantities  of  rela- 
tively pure  pigment.  With  the  facilities  available  in  most  laboratories 
10  kgs.  of  dried,  highly  pigmented  leaves  would  be  somewhat  burden- 
some to  carry  through  rapidly  enough  to  avoid  loss  of  pigment  by 
oxidation.  The  yield  of  relatively  pure  pigment  by  this  method  would 
not  be  over  0.5  grams  at  the  most,  using  highly  pigmented  spinach 
leaves  as  the  source  of  material. 

The  final  purification  of  carotin  is  carried  out  in  the  usual  manner, 
namely,  by  repeated  precipitations  from  carbon  disulfide  by  absolute 
alcohol  and  a  final  crystallization  from  the  lowest  boiling  petroleum 
ether.  The  final  yield  of  perfectly  pure  pigment  would  naturally  be 
somewhat  less  than  the  figures  mentioned  above. 

The  most  tedious  features  of  this  process  are  the  removal  of  the 
small  amount  of  extracted  chlorophyll  from  the  dried  leaves,  and  the 
final  purification  of  the  crude  carotin.  It  is  not  likely  that  the  frac- 
tional precipitations  and  recrystallizations  can  well  be  avoided.  It 
seems  feasible,  however,  to  substitute  a  more  direct  method  for  remov- 
ing the  chlorophyll.  Tswett  (1906b)  has  shown  that  if  a  petroleum 
ether  solution  of  chlorophyll  and  carotin  is  shaken  with  an  excess  of 
dry,  finely  divided  CaCOs,  inulin  or  sucrose,  the  chlorophyll  is  com- 


204  CAROTINOIDS  AND  RELATED  PIGMENTS 

pletely  adsorbed,  leaving  the  carotin  in  solution.  Any  carotin  held 
mechanically  by  the  adsorbing  material  can  be  washed  out  with 
petroleum  ether  without  removing  the  adsorbed  chlorophyll.  It  would 
seem  entirely  practicable  to  apply  these  facts  to  the  isolation  of  crys- 
talline carotin  from  leaves.  The  removal  of  the  chlorophyll  could  be 
postponed  until  the  bulk  of  the  petroleum  ether  was  distilled  off  and 
before  the  final  concentration  and  crystallization  of  the  pigment. 
According  to  the  laws  of  adsorption  it  may  be  expected  that  the  ad- 
sorbing material  will  also  remove  h  certain  amount  of  some  of  the 
other  impurities.  In  applying  this  method  care  must  be  taken  to 
choose  only  the  most  finely  divided  adsorbing  agent. 

The  problem  of  securing  pigment  solutions  from  fresh  or  dried  plant 
tissues  merely  for  macroscopic  examination  is  much  less  compli- 
cated. Fresh  tissues  should  first  be  macerated.  Tswett  recom- 
mends the  use  of  a  little  CaCOg  or  MgO  in  connection  with  the 
maceration  to  neutralize  the  acids  in  the  plant  sap.  In  order  to 
choose  the  proper  solvent  it  is  well  to  have  in  mind  certain  rules 
laid  down  by  Tswett  (1906b)  for  the  action  of  the  various  solvents 
upon  the  carotinoid  and  chlorophyll  pigments  in  plants.  According 
to  Tswett  the  solvents  commonly  used  are  divided  into  three  groups 
according  to  their  relations  toward  the  leaf  pigments. 

1.  Alcohol  (methyl,  ethyl,  amyl),  acetone,  acetaldehyde,  ether, 
chloroform. — These  solvents  acting  on  fresh  (macerated)  or  dried 
leaves  dissolve  out  all  the  pigments  equally  and  completely. 

2.  Petroleum  ether  and  petroleum  benzine  (low  or  high  b.  p.  petro- 
leum ether). — Fresh  leaves  (macerated)  give  more  or  less  yellow 
extracts  when  treated  with  these  solvents.  The  chief  pigment  is  carotin 
but  traces  of  other  pigments  are  also  extracted.  Leaves  dried  at 
low  temperature  likewise  give  up  their  carotin  to  these  solvents,  and 
in  somewhat  purer  condition.  Plant  tissues  which  have  been  cooked, 
or  only  warmed  to  a  moderately  high  temperature,  however,  give 
green  extracts  when  macerated  with  these  solvents. 

3.  Benzene,  xylene,  toluene  and  carbon  disulfide. — ^These  solvents 
act  intermediately  between  the  first  and  second  groups.  For  the 
extraction  of  all  the  chlorophyll  and  carotinoid  pigments  Tswett  recom- 
mends petroleum  ether  containing  10  per  cent  absolute  alcohol  for 
fresh  leaves  and  petroleum  ether  containing  1  per  cent  alcohol  for 
dry  leaves. 

Animal  fat.  It  is  manifestly  impossible  to  secure  carotin  in  appre- 
ciable quantities  from  animal  fat,  like  butter  fat,  or  from  the  highly 


1 


METHODS  OF  ISOLATION  OF  CAROTINOIDS         205 

pigmented  adipose  tissue  which  one  finds  in  certain  breeds  of  dairy 
cattle.  At  the  most  butter  fat  contains  little  more  than  0.005  per 
cent  carotin,  and  in  many  cases  considerably  less  than  this  amount. 
Ten  to  20  kgs.  of  fat  would  therefore  be  required  to  secure  0.5  grams 
of  pigment,  assuming  that  all  of  it  could  be  recovered.  The  problem 
is  rendered  still  more  difficult  by  the  fact  that  the  fat  must  be  com- 
pletely saponified  before  the  pigment  can  be  extracted;  and,  if  it  is 
necessary  to  use  a  procedure  in  connection  with  the  saponification 
and  extraction  of  pigment  from  large  quantities  of  fat  such  as  has 
been  found  practicable  for  small  quantities,  the  volumes  of  soap 
solution  and  ether  required  for  the  operation  would  soon  reach  a 
magnitude  all  out  of  proportion  to  the  facilities  of  the  best  appointed 
laboratories.  To  be  specific,  at  least  120  liters  of  ether  would  be 
necessary  to  secure  the  carotin  from  10,000  grams  of  fat,  and  inas- 
much as  the  yield  could  not  be  over  a  few  tenths  of  a  gram  of  crystal- 
line product  the  mechanical  difficulties  involved  would  not  justify 
the  attempt. 

It  is  readily  possible,  however,  to  obtain  sufiicient  carotin  from 
animal  fat  for  a  macroscopic  study  of  the  chemical  and  physical 
properties  of  the  pigment.  Twenty  to  thirty  grams  of  well  colored 
butter  fat  or  rendered  adipose  tissue  fat  are  ample  for  such  a  study. 
The  butter  fat  must  •  not  be  artificially  colored,  the  pure  rendered 
butter  fat  from  Jersey  or  Guernsey  cows  on  a  fresh  pasture-grass 
diet  being  best  suited  for  the  experiment.  The  fat  must  first  be 
saponified;  and,  in  this  connection,  an  important  precaution  must 
be  taken,  namely,  to  avoid  the  use  of  alcohol  which  has  not  been 
completely  purified  from  aldehydes  which  produce  yellow  to  red  col- 
ored resins  with  alkali.^  The  resins  thus  formed  follow  the  caro- 
tinoids  in  their  isolation  and  interfere  greatly  with  the  study  of  the 
properties  of  the  pigments. 

For  the  saponification  of  the  fat,  2  cc.  of  colorless  20  per  cent 
alcoholic  potash  is  added  for  each  gram  of  fat  and  the  mixture  al- 
lowed to  boil  for  about  one  hour  under  a  reflux  condenser.     The 

'  Ethyl  alcohol  is  especially  likely  to  contain  such  impurities.  It  can  be  purified  best 
by  treatment  with  silver  nitrate,  in  which  about  2.0  grams  of  crystals  are  added  to  4 
liters  of  alcohol  and  allowed  to  stand,  with  shaking,  for  several  days.  200  g.  unslaked 
lime  are  now  added  to  precipitate  the  AgO,  neutralize  the  acids  and  remove  any  excess 
water.  The  lime  can  now  be  filtered  off  and  the  filtrate  distilled.  Usually  one  such 
treatment  will  prepare  an  excellent  98  per  cent  alcohol  which  will  show  no  coloration 
on  boiling  in  the  presence  of  20  per  cent  KOH.  Should  a  color  develop  under  these 
conditions  the  purification  must  be  repeated. 

Methyl  alcohol  can  also'  be  used  for  the  saponification  of  the  fat,  but  it,  also,  must 
show  no  coloration  when  a  20  per  cent  KOH  solution  of  the  alcohol  is  boiled. 


206  CAROTINOIDS  AND  RELATED  PIGMENTS 

resulting  soap  is  dissolved  in  three  volumes  of  distilled  water.  After 
cooling,  this  solution  is  shaken  with  an  equal  volume  of  pure  ether 
in  a  separatory  funnel.  The  extraction  is  repeated  with  a  fresh 
volume  of  ether  equal  to  one-half  the  volume  of  soap  solution.  The 
soap  should  now  be  colorless.^  The  combined  ether  extracts  are 
now  washed  many  times  with  an  excess  of  water,  carefully  at  first 
to  avoid  emulsions,  and  more  vigorously  with  subsequent  washings. 
When  the  wash  water  no  longer  reacts  alkaline  to  phenolphthalein, 
the  ether  solution  is  dried  by  shaking  with  neutral,  fused  CaClg  or 
anhydrous  NagSO^  for  a  few  hours,  decanted  or  filtered  from  the 
inorganic  drying  agents  and  evaporated  to  dryness  in  a  dry  vacuum. 
Little  or  no  heat  need  be  applied  because  of  the  rapid  volatilization  of 
ether  under  diminished  pressure.  The  residue  consists  of  pigment 
mixed  with  large  quantities  of  cholesterol  and  traces  of  other  unsaponi- 
fiable  matter.  According  to  Steenbock  (1921a)  and  others,  the  fat- 
soluble  vitamine  in  butter  fat  is  present  in  this  fraction.  The  choles- 
terol can  be  removed  by  the  digitonin  method  of  Windaus  (1909), 
by  dissolving  the  residue  in  warm  95  per  cent  alcohol  and  adding 
an  excess  of  a  hot  one  per  cent  solution  of  digitonin  in  90  per  cent 
alcohol.  This  procedure  is  not  necessary,  however,  for  the  study  of 
the  chemical  and  physical  properties  of  the  pigment. 

The  examination  of  pigment  isolated  from  animal  fat  in  the  above 
manner  must  be  made  at  once  unless  facilities  are  available  for  keep- 
ing the  pigment  in  an  atmosphere  of  inert  gas.  Kohl  (1902b)  states 
that  crystalline  carotin  can  be  protected  completely  from  oxidation, 
even  in  the  sunlight,  if  placed  under  glycerin.  The  writer  has  never 
tried  this  method  for  crude  preparations  of  pigment  from  animal 
tissues,  so  is  unable  to  vouch  for  its  usefulness  for  pigments  prepared 
by  the  method  just  given. 

Blood  serum.  The  blood  serum  of  man  and  certain  animals  may 
be  relatively  rich  in  carotin,  giving  it  a  golden  yellow  color.  While 
this  material  can  not  be  expected  to  serve  as  a  suitable  source  of 
pigment  in  large  quantities  the  pigment  can  be  isolated  in  sufficient 
amounts  for  chemical  examination  without  great  difiiculty.  Serum 
or  plasma  free  from  erythrocytes  must  first  be  obtained.  This  may 
be  done  either  by  allowing  the  blood  to  clot  and  permitting  the  serum 

*  Many  of  the  early  workers  who  saponified  their  plant  or  animal  extracts  evaporated 
the  alcohol  and  extracted  the  dried  soaps  with  the  solvents,  or  carried  out  the  extrac- 
tions with  soaps  which  had  been  salted  out  of  aqueous  solution  with  NaCl.  In  the 
writer's  experience  these  procedures  are  not  advantageous  When  working  with  pure 
animal  fats  which  contain  carotinoids. 


METHODS  OF  ISOLATION  OF  CAROTINOIDS         207 

to  separate  when  the  clot  contracts,  or  by  defibrinating  the  freshly 
drawn  blood  by  whipping  it  vigorously,  filtering  off  the  fibrin  and 
centrifuging  the  erythrocytes  from  the  defibrinated  plasma,  or  merely 
by  drawing  the  blood  into  sufficient  saturated  potassium  oxalate  or 
sodium  citrate  solution  to  prevent  clotting  and  throwing  down  the 
erythrocytes  from  the  oxalated  or  citrated  blood  with  the  centrifuge. 
Each  of  the  three  preparations,  namely,  serum,  defibrinated  plasma 
or  oxalated  (or  citrated)  plasma  serve  equally  well  for  the  isolation 
of  the  serum  carotinoids. 

In  most  cases  carotin,  when  present  in  blood,  appears  to  be  in 
some  sort  of  physico-chemical  combination  with  a  fraction  of  the 
albumin  in  colloidal  solution  in  the  blood.  Whatever  the  expla- 
nation of  the  state  of  the  pigment  in  the  blood  may  be  in  these 
cases,  the  fact  remains  that  when  this  occurs  the  direct  extraction 
of  the  pigment  with  ether,  petroleum  ether,  chloroform,  carbon  disul- 
fide or  any  of  the  usual  carotin  solvents  is  impossible.  However,  if 
the  serum  is  first  treated  with  an  equal  volume  of  alcohol,  the 
carotin  can  be  readily  extracted  by  shaking  with  the  solvents  men- 
tioned. Based  on  this  fact  the  writer  devised  the  following  method 
for  extracting  the  carotin  from  blood  serum:  Clear  serum  or  plasma 
is  mixed  with  an  excess  of  plaster  of  Paris,  using  about  40  grams 
of  the  CaSO^  for  each  10  cc.  of  serum.  The  damp  powder  is  trans- 
ferred to  a  flask,  alcohol  added  equal  to  the  volume  of  serum  and 
thoroughly  mixed  with  the  plaster  of  Paris  mass.  An  equal  volume 
of  low  boiling  petroleum  ether  is  now  added  and  vigorously  shaken 
with  the  mass.  On  standing,  the  petroleum  ether  rises  to  the  surface, 
giving  an  almost  quantitative  extraction  of  the  carotin.  The  extract 
can  be  readily  poured  off  and  the  extraction  repeated  with  fresh 
petroleum  ether  in  order  to  insure  a  complete  extraction. 

Reference  has  already  been  made  to  the  manner  in  which  blood 
carries  the  carotin.  Until  recently  the  writer  held  the  view  that 
carotin  is  always  present  in  some  sort  of  combination  with  an  albumin 
fraction  in  the  serum.  So  far  as  his  experience  with  the  blood  of 
cattle  and  horses  is  concerned  this  view  still  holds.  However,  he  has 
recently  examined  the  blood  of  several  diabetics  on  vegetarian  diets 
containing  much  green  food  in  which  this  carotin-albumin  com- 
bination did  not  appear  to  exist.  At  least  the  pigment,  which  proved 
to  be  carotin,  or  at  any  rate  to  have  the  relative  solubility  and  other 
chemical  properties  of  carotin,  and  not  xanthophyll,  was  readily  and 
completely  extracted  from  the  serum  merely  by  vigorous  shaking  with 


208  CAROTINOIDS  AND  RELATED  PIGMENTS 

fresh  portions  of  pure  ether.  Even  in  these  cases  extraction  of  the 
pigment  could  be  facilitated  by  diluting  the  serum  with  two  volumes 
of  water  and  adding  an  equal  volume  of  methyl  alcohol  before  shaking 
with  ether,  . 

In  the  writer's  experience  with  cattle  and  horse  serum  the  evidence 
for  a  carotin-albumin  combination  of  some  kind  rests  upon  a  number 
of  easily  demonstrated  facts,  among  which  are  the  following.  The 
fat  solvents  will  extract  little  if  any  pigment  from  serum  even  after 
great  dilution  with  water.  When  the  globulins  and  albumins  in  the 
serum  are  fractionally  precipitated  by  increasing  concentrations  of 
ammonium  sulfate,  the  carotin  follows  the  albumin  fractions.  In 
fact  it  is  possible  to  roughly  isolate  an  albumin  which  carries  the 
carotin  in  firm  combination,  which,  like  the  serum  itself,  will  not 
give  up  its  pigment  to  the  fat  solvents  until  first  treated  with  alco- 
hol, indeed  unless  alcohol  is  present.  The  lead,  silver  and  mercury 
salts  of  the  protein  act  in  the  same  manner.  After  coagulation  with 
alcohol  and  drying  it  was  found,  in  one  test  at  least,  that  alcohol  had 
to  be  added  before  petroleum  ether  would  extract  the  pigment  from 
the  protein.  This  albumin,  moreover,  seems  to  have  a  more  or  less 
definite  heat-coagulation  point  of  86°  C,  when  in  half-saturated  am- 
monium sulfate  solution.  This  property  can  therefore  be  used  for 
the  isolation  of  the  pigment-carrying  protein. 

The  isolation  of  the  carotin-albumin  complex  can  be  carried  out 
as  follows: — The  serum  is  first  freed  from  globulins  by  adding  an 
equal  volume  of  saturated  ammonium  sulfate  solution.  These  are 
filtered  off  on  a  Biichner  funnel,  using  suction,  and  thoroughly  washed 
with  half  saturated  ammonium  sulfate  solution.  The  combined  filtrate 
and  washings  are  then  carefully  heated  to  a  temperature  of  79°  C, 
at  which  temperature  the  bulk  of  the  albumins  are  coagulated. 
Some  carotin  is  lost  in  this  coagulum,  but  with  serum  rich  in  carotin 
the  filtrate  from  these  proteins  will  have  a  golden  yellow  color.  The 
carotin-albumin  fraction  is  secured  from  this  filtrate  either  by  salting 
it  out  by  any  of  the  albumin  precipitants  (complete  saturation  with 
ammonium  sulfate  is  best)  or  by  heating  to  86°  C.  In  either  case 
the  precipitate  will  have  a  deep  yellow  color  and  the  amount  ob- 
tained will  be  very  small  in  comparison  with  the  proteins  which 
have  been  precipitated  as  globulins  and  albumins  in  the  preliminary 
operations.  The  protein  can  be  redissolved  after  salting  out,  and  the 
aqueous  solution  thus  obtained  exhibits  all  the  properties  of  blood 
serum  so  far  as  its  relations  to  fat  solvents  and  the  extraction  of 


METHODS  OF  ISOLATION  OF  CAROTINOIDS         209 

the  pigment  are  concerned.  The  writer  has  long  been  impressed  with 
the  possibility  of  throwing  light  upon  the  formation  of  milk  fat 
through  a  study  of  this  interesting  complex  in  the  blood  of  cattle, 
inasmuch  as  it  is  unquestionably  the  source  from  which  the  milk 
fat  derives  its  natural  pigment.  The  failure  of  the  fat  solvents  to 
remove  the  pigment  from  this  protein  complex  by  direct  extraction 
indicates  that  the  pigment  becomes  a  part  of  the  milk  fat  through 
a  process  much  more  deep-seated  than  a  mere  solvent  action.  It 
seems  very  probable,  therefore,  that  this  caroto-albumin  plays  some 
important  part  in  the  process  of  fat  synthesis  in  the  mammary  gland 
of  the  cow. 

Isolation  of  Xanthophylls 

Green  leaves.  It  was  clearly  shown  in  Chapter  II  that  a  group  of 
xanthophyll  pigments  accompany  chlorophyll  and  carotin  in  green 
leaves.  It  is  not  known,  however,  whether  the  crystalline  xanthophyll 
which  can  be  isolated  from  the  green  leaves  of  plants  is  a  mixture 
of  the  xanthophyll  isomers  or  consists  of  the  major  xanthophyll 
constituent,  the  xanthophyll  a  of  Tswett.  The  evidence  on  both  sides 
of  the  question  was  presented  in  Chapter  11. 

Willstatter  and  Mieg  (1907)  were  the  first  to  isolate  crystalline 
xanthophyll  in  quantity.  Their  method  was  as  follows.  Air-dried, 
powdered  nettle  leaves  were  extracted  with  cold  95  per  cent  alcohol, 
which  extracted  the  chlorophylls  and  xanthophylls,  but  very  little 
of  the  carotin.  The  extract  was  treated  in  the  cold  with  KOH,  con- 
verting the  chlorophylls  into  chlorophyllins,  which  precipitated  in 
part  directly  from  the  alcoholic  solution  and  partly  on  addition  of 
much  ether.  The  alkaline  alcoholic-ether  solution  was  shaken  with 
successive  portions  of  fresh  water  until  all  the  green  color  was  re- 
moved from  the  ether  layer.  The  combined  ether  solutions  obtained 
in  this  way  from  100  kgs.  of  dried  nettle  leaves  were  concentrated  to 
a  volume  of  6  liters  and  after  more  washing  with  alcoholic  potash  and 
water  and  drying  with  anhydrous  sodium  sulfate,  were  mixed  with 
two  volumes  of  petroleum  ether.  This  precipitated  the  xanthophyll 
and  a  considerable  amount  of  colorless,  high  molecular  weight  alcohol, 
the  xanthophyll  coming  down  as  a  reddish-yellow  precipitate. 

To  remove  the  impurities  from  the  precipitated  xanthophyll  Will- 
statter and  Mieg  boiled  the  precipitate  with  1200  cc.  of  acetone,  which 
left  a  part  of  the  colorless  substance  undissolved.  The  warm  acetone 
solution  was  treated  with  about  two  volumes  of  methyl  alcohol.     In 


210  CAROTINOIDS  AND  RELATED  PIGMENTS 

the  course  of  two  days  the  xanthophyll  crystallized  out  at  room  tem- 
perature as  yellow  to  orange-red  tablets  with  a  brilliant  steel-blue 
reflection.  Willstatter  and  Mieg  secured  a  yield  of  12  grams  of  crude 
crystals  from  the  100  kgs.  of  dried  nettle  leaves.  Further  purification 
was  obtained  by  crystallization  from  boiling  methyl  alcohol,  from 
which  the  crystals  come  down  with  one  molecule  of  alcohol  of  crystal- 
lization, or  by  dissolving  in  the  least  possible  amount  of  chloroform 
and  adding  an  excess  of  petroleum  ether,  in  which  the  xanthophyll 
is  almost  insoluble. 

Jorgensen  and  Stiles  (1917)  have  described  what  appears  to  be  a 
more  convenient  method  for  isolating  crystalline  xanthophyll,  which 
also  gives  higher  yields  than  the  method  of  Willstatter  and  Mieg. 
They  prefer  the  dry,  powdered  nettle  leaves  because  of  their  excellent 
keeping  quality,  but  in  this  respect  spinach,  which  is  richer  in  caro- 
tinoids,  should  serve  equally  well  as  a  source  of  pigment. 

In  Jorgensen  and  Stiles'  method  the  air  dried,  powdered  leaves 
are  submitted  to  a  final  drying  in  vacuum,  over  H2SO4.  About  500 
grams  of  this  powder  are  placed  on  a  filter  paper  in  a  Biichner  funnel 
24  cm.  in  diameter  and  sucked  to  the  paper  with  a  strong  water  pump 
or  vacuum  pump.  To  get  the  best  results  the  powder  must  be  thor- 
oughly dry  and  be  sucked  in  a  coherent  mass  not  more  than  5  cm. 
deep  on  the  funnel.  Half  a  liter  of  80  per  cent  acetone  is  now  al- 
lowed to  permeate  the  powder  on  the  filter  for  5  minutes  without  the 
use  of  the  pump.  Then  250  cc.  of  solvent  are  added  and  slowly  sucked 
through  with  the  pump.  After  5  minutes  another  250  cc.  portion  of 
solvent  is  added  and  sucked  through  with  the  pump  for  10  minutes. 
This  operation  is  repeated  with  two  further  250  cc.  portions  of  80  per 
cent  acetone,  and  finally  the  pump  is  allowed  to  act  as  strongly  as 
possible  until  the  powder' is  sucked  dry.  The  1,500  cc.  of  solvent 
used  give  800  to  900  cc.  of  extract. 

When  the  extract  has  been  obtained  from  2  kgs.  of  dry  powder  m 
this  manner,  the  fractions  are  combined  and  washed  free  from  many 
impurities  and  finally  from  acetone  by  the  following  procedure.  The 
acetone  solution  is  added  in  two  successive  portions  to  4  liters  of 
petroleum  ether  (sp.  g.  0.64  to  0.66)  in  a  separatory  funnel  of  7  liter 
capacity.  Water  (0.5  liters)  is  added  with  each  of  these  additions 
while  the  funnel  is  being  gently  rotated,  and  after  separation  into 
two  layers  the  lower  layer  is  drawn  off  and  discarded  before  the 
addition  of  the  second  portion.  The  petroleum  ether  layer  is  now 
mixed  with  two  successive  liters  of  80  per  cent  acetone  solution,  the 


METHODS  OF  ISOLATION  OF  CAROTINOIDS         211 

acetone  being  removed  each  time  by  adding  4  successive  liters  of 
water  with  gentle  rotation  of  the  liquid  and  drawing  off  the  lower  layer 
each  time. 

The  petroleum  ether  solution  now  remaining  contains  the  xantho- 
phylls,  the  chlorophylls  and  the  carotin.  The  xanthophylls,  with  some 
chlorophyll,  are  removed  by  shaking  with  three  successive  additions 
of  2  liters  of  80  per  cent  methyl  alcohol.  After  each  addition  and 
shaking  the  methyl  alcohol  layer  is  removed.  If  the  last  extract  is 
still  considerably  yellow  additional  extractions  are  made  until  the 
alcohol  layer  is  practically  colorless.  The  xanthophyll  in  the  com- 
bined methyl  alcohol  extracts  is  next  freed  from  chlorophyll  by  trans- 
ferring to  ether  in  the  following  manner:  4  to  5  liters  of  ether, 
a  quantity  of  water  and  30-50  cc.  of  concentrated  methyl  alcoholic 
potash  are  added,  and  the  mixture  shaken.  The  liquids  are  allowed 
to  separate,  the  lower  layer  is  drawn  off  and  discarded  and  the  ether 
washed  with  water  until  no  more  green  color  is  extracted.  The  ether 
is  now  dried  with  anhydrous  Na2S04,  evaporated  in  vacuum  to  a 
volume  of  30  cc,  200  to  300  cc.  of  methyl  alcohol  added,  and  the 
ether  removed  completely  by  further  concentration  in  vacuum. 
Xanthophyll  precipitates  out  on  cooling  the  hot,  concentrated  methyl 
alcohol,  the  addition  of  a  little  water  helping  the  precipitation.  The 
yield  of  crude  xanthophyll  by  this  method  is  stated  by  Jorgensen  and 
Stiles  to  be  0.8  grams  from  2  kgs.  of  dried  nettle  leaves  which  is 
over  three  times  as  much  as  Willstatter  and  Mieg  secured  by  their 
method.  The  method  just  described  has  the  additional  advantage  that 
the  xanthophyll-free  petroleum  ether  can  be  used  for  the  isolation 
of  carotin.^ 

The  foregoing  methods  are  best  suited  for  the  isolation  of  crystal- 
line xanthophyll  in  quantity.  A  solution  of  mixed  xanthophylls  for 
macroscopic  examination  can  be  secured  by  the  following  simple  pro- 
cedure.    About  25  grams  of  dried  powdered  leaves  or  fresh  leaves 

» The  method  recommended  is  to  wash  the  petroleum  ether,  now  consisting  of  about 
3.5  liters,  four  times  with  two  liter  portions  of  water  to  remove  the  last  traces  of 
acetone  and  methyl  alcohol.  As  the  last  traces  of  these  solvents  are  removed  the 
chlorophyll  present  in  the  petroleum  ether  precipitates  as  a  fine  suspension.  A  little 
anhydrous  Na2S04  is  added  to  take  up  the  water  and  then  150  grams  of  CaCOa,  and 
the  solution  finally  filtered  through  a  layer  of  CaCOg  on  a  Biichner  funnel.  This  treat- 
ment takes  out  the  chlorophyll  suspension.  The  filtrate  is  evaporated  in  vacuum  at 
40°  C.  and  the  oily  residue  treated  with  300  cc.  of  90  per  cent  alcohol.  The  carotin 
begins  to  crystallize  out  immediately  and  is  complete  on  standing  in  the  cold.  Purifica- 
tion is  effected  by  shaking  up  the  crystalline  mass  with  200-300  cc.  of  petroleum  ether 
and  filtering  quickly  and  repeating  the  washing  with  a  mixture  of  two  parts  of 
petroleum  ether  and  one  part  of  absolute  alcohol.  The  yield  of  0.25  grams  from  two 
kgs.  of  dried  nettle  leaves  is  much  greater  than  Willstatter  and  Mieg  secured. 


212  CAROTINOIDS  AND  RELATED  PIGMENTS 

which  have  been  macerated  with  emery  in  the  presence  of  CaCOg  or 
MgO  (to  neutralize  plant  acids)  are  allowed  to  stand  in  contact  with 
pure  carbon  disulfide  in  a  stoppered  flask  for  24-48  hours.  The 
solvent  is  filtered  off  and  evaporated  to  dryness  in  vacuum.  The 
residue  is  boiled  for  thirty  minutes  with  50  cc.  of  20  per  cent  methyl 
or  ethyl  alcoholic  potash  (using  only  solutions  which  alone  give  no 
coloration  whatever  on  boiling).  After  cooling,  150  cc.  of  distilled 
water  are  added  and  the  mixture  shaken  with  200  cc.  of  pure  ether 
in  a  separatory  funnel.  After  the  two  layers  have  separated  the  lower 
greenish  layer  is  drawn  off  and  shaken  with  100  cc.  of  fresh  ether. 
A  third  extraction  with  fresh  ether  should  not  be  necessary,  but  can 
be  tried  to  insure  the  complete  extraction  of  the  carotinoids.  The 
combined  golden  yellow  ether  extracts,  which  may  have  a  slight  green 
tinge,  are  washed  with  successive  equal  portions  of  distilled  water 
until  the  washings  no  longer  react  alkaline  to  phenolphthalein.  The 
ether  may  now  be  filtered  through  a  layer  of  powdered  anhydrous 
Na2S04,  to  remove  the  water.  The  filtrate  is  evaporated  to  dryness 
in  vacuum  and  the  residue  taken  up  at  once  in  100  cc.  of  hot  petro- 
leum ether  (b.  p.  30-50°  C).  After  cooling,  the  solution  is  shaken 
with  successive  100  cc.  portions  of  80  per  cent  methyl  alcohol  until 
no  more  color  is  extracted.  The  combined  methyl  alcohol  solutions 
contain  the  xanthophylls.  On  dilution  with  water  to  form  a  25-30 
per  cent  alcohol  solution  ether  will  now  extract  these  pigments.  After 
washing  the  ether  free  from  alcohol  with  water  and  drying  with 
Na2S04,  the  ether  can  be  evaporated  off  in  vacuum  and  the  pig-: 
mented  residue  used  for  an  examination  of  any  of  the  usual  xantho-: 
phyll  properties. 

Egg  yolk.  The  large  amount  of  protein,  fat,  lecithin  and  other 
lipoids  in  egg  yolk  presents  certain  rather  difficult  problems  in  the 
isolation  of  the  xanthophyll  pigment  present.  The  isolation  was  ac- 
complished, however,  by  Willstatter  and  Escher  (1912)  in  the  fol- 
lowing manner,  but  not  without  loss  of  a  great  deal  of  pigment,  as  can 
be  readily  seen.  Egg  yolk  weighing  100  kgs.,  representing  6,000 
eggs,  was  beaten  up  and  6  kg.  portions  placed  in  stone  jars  with  7 
liters  of  methyl  alcohol  to  coagulate  the  protein.  The  coagulum  was 
separated  by  means  of  the  centrifuge,  the  alcohol,  it  is  stated,  being 
almost  free  from  color.  Each  portion  of  coagulum,  amounting  to  a 
little  over  5  kgs.,  was  thoroughly  mixed  with  3  liters  of  acetone,  and 
the  golden  yellow  extract  sucked  off  through  a  sand  filter.  After 
the  coagulum  from  each  6  kg.  portion  of  egg  yolk  had  been  extracted 


METHODS  OF  ISOLATION  OF  CAROTINOIDS         213 

in  this  way  the  residue  (94  kgs.  in  all)  was  divided  into  portions  of 
2.8  kgs.  and  each  portion  shaken  twice  with  fresh  two  liter  quan- 
tities of  acetone  in  a  shaking  machine  for  one  hour,  the  acetone  being 
sucked  off  each  time  through  a  sand  filter.  Practically  all  the  color 
was  extracted  by  this  means. 

All  the  acetone  extracts  were  now  combined  and  amounted  to  200 
liters.  About  2.25  liters  of  oil  settled  out  on  standing.  Although 
highly  colored  it  was  discarded.  The  next  problem  was  to  remove  the 
phosphatides  and  cholesterol.  The  phosphatides  were  removed  by 
mixing  each  6  liter  portion  of  acetone  extract  with  0.5  liter  of  petro- 
leum ether  (sp.  g.,  0.64-0.66),  and  adding  three  volumes  of  water 
carefully,  to  avoid  an  emulsion.  The  lower  watery  acetone  layer  was 
drawn  off  after  standing  a  day  and  the  dark  brown,  thick  oily  syrup 
rinsed  out  with  petroleum  ether.  Twenty  liters,  in  all,  of  this  oily 
material  were  obtained.  Large  clumps  of  almost  colorless  phospha- 
tides, amounting  to  nearly  2  kgs.  were  thrown  down  by  adding  2 
volumes  of  acetone  to  the  petroleum  ether  solution  of  this  syrup.  The 
pigmented  acetone-petroleum  ether  solution  was  decanted,  filtered 
through  linen,  and  freed  from  acetone  again  by  washing  with  water, 
first  by  decantation  and  finally  by  direct  addition  of  water,  allowing 
about  one  hour  between  each  addition.  The  reddish-brown  petroleum 
ether  solution  was  now  filtered  through  fused  Na2S04  and  the  filtrate 
concentrated  to  2  liters  at  30-35°  C,  in  vacuum,  i.  e.,  until  the  syrup 
set  to  a  crystalline  mass  of  cholesterol.  This  was  filtered  off  and  the 
deep  colored  filtrate  diluted  with  4  liters  of  petroleum  ether  (b.  p. 
30°-50°  C).  On  standing  in  the  ice  box  for  a  few  days  most  of  the 
pigment  crystallized  out  as  a  bright  red  blanket  of  very  fine  needles. 
The  yield  of  crude  pigment  amounted  to  4  grams.  The  purification 
of  the  pigment  was  described  in  Chapter  VI.  It  is  of  interest  that 
the  method  of  isolation  used  by  Willstatter  and  Escher  shows  that 
a  portion,  at  least_,  of  the  egg  yolk  carotinoids  are  not  present  dis- 
solved in  fat.  It  was  not  found  necessary  to  resort  to  a  saponification 
of  the  extracts  in  order  to  isolate  crystals  of  pigment. 

The  separation  of  sufficient  egg  yolk  pigment  for  macroscopic  ex- 
amination can  be  effected  in  a  satisfactory  manner  from  a  single  well- 
colored  egg  yolk.  For  this  purpose  the  following  procedure  gives 
very  satisfactory  results.  The  raw  yolk  is  thrown  into  100  cc.  of 
acetone,  and,  after  heating  to  boiling,  filtered  to  remove  the  coagu- 
lated, colorless  proteins.  The  filtrate  is  evaporated  and  the  residue 
saponified  with  50  cc.  of  20  per  cent  methyl  alcoholic  potash  solution 


214  CAROTINOIDS  AND  RELATED  PIGMENTS 

at  boiling  temperature  for  about  one  hour,  taking  care  to  use  alco- 
holic potash  which  itself  gives  rise  to  no  color  on  heating.  The 
pigment  is  extracted  from  the  saponified  material  using  the  pro- 
cedure given  for  isolating  the  pigment  of  butter  fat.  The  ether  solu- 
tion of  pigment  is  dried  by  filtering  through  a  layer  of  anhydrous 
NagSO^  and  evaporated  to  dryness  in  vacuum.  The  chief  impurity 
in  the  residue  will  be  cholesterol.  By  dissolving  in  the  least  possible 
amount  of  hot  methyl  alcohol  and  cooling  to  a  low  temperature  a 
great  deal  of  the  cholesterol  will  precipitate  out  and  can  be  removed 
by  filtration.  The  cholesterol  which  remains  will  not  interfere  with 
the  examination  of  the  pigment.  In  the  writer's  experience  egg  yolk 
pigment  prepared  in  this  way  will  invariably  show  the  presence  of 
a  small  amount  of  pigment  which  cannot  be  extracted  from  petro- 
leum ether  by  80  per  cent  methyl  alcohol,  indicating  that  carotin-like 
pigments  are  not  entirely  absent  from  egg  yolk. 

Blood  serum.  It  is  not  necessary  to  dwell  at  length  on  the  isola- 
tion of  xanthophyll  from  blood  serum  in  view  of  the  detailed  descrip- 
tion already  given  of  the  procedure  to  be  used  for  isolating  carotin 
from  blood.  One  or  two  points,  however,  should  be  emphasized. 
Xanthophylls  are  found  most  abundantly  in  the  blood  serum  of  fowls, 
as  has  already  been  pointed  out.  This  does  not  mean,  however,  that 
blood  rich  in  carotin,  like  cattle  blood,  is  necessarily  devoid  of  xantho- 
phylls. In  order  to  show  the  presence  of  these  pigments  in  cattle 
blood  it  is  necessary  to  extract  200-300  cc.  of  desiccated  (with  CaS04) 
serum  completely  with  ether  as  well  as  with  petroleum  ether,  after 
treating  with  alcohol.  The  combined  pigments  from  well  colored 
serum  will  show  the  presence  of  xanthophylls  when  submitted  to  the 
phase  test  or  analyzed  by  means  of  the  chromatograph. 

It  has  been  the  writer's  invariable  experience  with  the  blood  of  fowls 
that  the  xanthophylls  present  can  be  readily  extracted  by  direct  shak- 
ing of  either  the  fresh  or  desiccated  serum  with  ether.  The  experience 
of  van  den  Bergh  and  Muller  (1920)  has  been  contrary  to  this,  these 
investigators  finding  a  number  of  cases  in  which  ether  extraction 
failed.  No  explanation  is  as  yet  apparent  for  this  divergence  in  our 
experiences.  However,  in  view  of  the  fact  that  it  appears  possible 
for  cases  to  occur  in  which  ether  extraction  alone  fails  to  remove  the 
pigment  the  writer  advises  that  desiccated  blood  serum,  in  which 
xanthophylls  are  suspected  to  exist,  be  extracted  first  with  ether, 
then  treated  with  alcohol  and  the  ether  extraction  repeated. 

Until  recently  the  writer  believed  that  the  direct  extraction  of  caro- 


METHODS  OF  ISOLATION  OF  CAROTINOIDS         215 

tinoid  from  blood  serum  by  ether  was  a  criterion  of  its  xanthophyll 
character.  The  instances  of  carotin  extraction  from  human  blood 
serum  which  the  writer  has  already  mentioned  show,  however,  that 
this  is  not  a  safe  basis  for  judging  the  character  of  the  pigment 
present. 

Isolation  of  Lycopin 

Several  investigators  have  described  the  isolation  of  lycopin,  which 
is  the  characteristic  red  pigment  of  tomatoes,  red  peppers,  the  pulp 
of  the  watermelon  and  a  number  of  tropical  fruits.  The  method  to 
be  described  here,  however,  is  that  used  by  Willstatter  and  Escher 
(1910),  who  first  showed  that  this  pigment  is  a  true  isomer  of  carotin. 
These  investigators  first  attempted  to  use  the  fresh  fruits  as  the 
source  of  pigment,  but  when  they  found  that  135  kgs.  of  tomatoes 
yielde(i  only  2.6  kgs.  of  dry  matter  from  which  only  2.7  grams  of 
crystalline  lycopin  could  be  obtained,  they  decided  on  a  canned 
preparation  of  concentrated  tomato  soup  of  Italian  make  as  better 
suited  for  their  work. 

Starting  with  74  kgs.  of  the  condensed  tomato  puree,  it  was  first 
dried  in  8  kg.  portions  by  shaking  with  4  liters  of  96  per  cent  alco- 
hol, collecting  the  coagulum  and  repeating  the  operation  with  two 
or  three  liters  more  of  the  alcohol.  The  coagulum  was  now  pressed 
as  dry  as  possible  and  finally  dried  completely  on  the  steam  bath 
before  grinding  to  a  powder.  The  total  yield  of  dry  powder  was  5.6 
kgs.  This  was  completely  extracted  with  carbon  disulfide  in  a  con- 
tinuous extractor  and  the  extract  evaporated  to  dryness  using  dimin- 
ished pressure  as  far  as  possible,  and  finally  at  a  temperature  of  40°  C. 
in  a  water  bath.  The  residue  was  now  treated  with  3  volumes  of 
absolute  alcohol,  transferred  to  a  suction  filter  and  washed  with  petro- 
leum ether.  The  yield  of  crude  pigment  amounted  to  11  grams.  The 
purification  of  the  pigment  was  accomplished  in  much  the  same  man- 
ner as  carotin  is  purified. 

Isolation  of  Fucoxanthin 

The  characteristic  algae  pigment,  fucoxanthin,  whose  chemical  rela- 
tion to  the  carotinoids  was  discovered  by  Willstatter  and  Page  (1914) 
was  isolated  in  quantity  by  them  in  the  following  manner.  Fifteen  to 
20  kgs.  of  the  fresh  algae  (Phaeophycece)  were  extracted  with  40  per 
cent  acetone,  using  2  liters  for  each  kg.  of  algae.    This  extract  was 


216  CAROTINOIDS  AND  RELATED  PIGMENTS 

discarded.  The  extracted  material  was  pulverized  and  extracted  at 
once  (i.  e.,  within  a  few  days)  with  5  portions  of  85  per  cent  alcohol. 
The  last  four  fractions  were  combined  (the  first  being  discarded)  and 
amounted  to  25  liters  for  the  20  kgs.  of  algae.  This  solution  was  next 
shaken  with  CaCOg  to  neutralize  acids,  and  decanted  from  the  set- 
tled chalk.  The  solution  was  now  diluted  with  water,  using  3.4 
volumes  for  each  10  volumes  of  extract.  The  chlorophyll  which  pre- 
cipitated was  allowed  to  settle  out  and  the  supernatant  fluid,  amount- 
ing to  40  liters  in  all,  used  as  the  mother  liquor  for  the  isolation  of  the 
fucoxanthin.    This  was  accomplished  as  follows: 

Four-liter  portions  were  treated  with  one  liter  of  a  mixture  of 
ether  and  petroleum  ether  (b.  p.,  30°-50°  C),  3:1,  and  1.5  liters  of 
water  added.  The  ether  layer  which  took  up  the  pigment  was  then 
washed  very  carefully  with  water  (to  avoid  emulsions)  in  order  to 
free  it  from  the  acetone  which  was  used  to  extract  the  pigments 
from  the  algse.  The  petroleum  ether  was  then  concentrated  to  0.5 
liter  in  vacuum,  diluted  with  an  equal  volume  of  ether  and  shaken 
with  70  per  cent  methyl  alcohol  saturated  with  petroleum  ether.  This 
removed  the  fucoxanthin,  together  with  some  xanthophyll.  The  xan- 
thophyll  was  removed  by  shaking  the  methyl  alcohol  with  an  equal 
volume  of  a  mixture  of  petroleum  ether  and  ether  (5:1).  The  fuco- 
xanthin was  then  transferred  to  ether,  the  ether  solution  was  concen- 
trated to  a  thick  syrup  and  about  1  liter  of  low  boiling  petroleum 
ether  added.  The  precipitate  of  crude  pigment  obtained  amounted  to 
about  2  grams  from  each  4  liter  portion  of  mother  liquor,  representing 
about  half  the  total  pigment  present.  The  crude  pigment  was  puri- 
fied by  recrystallization  from  methyl  alcohol,  giving  crystals  con- 
taining three  molecules  of  methyl  alcohol  of  crystallization,  which 
could  be  removed  in  vacuum.  Solvent-free  crystals  were  obtained 
by  precipitation  from  ether  with  low  b.  p.  petroleum  ether. 

Isolation  of  Rhodoxanthin 

This  pigment,  as  explained  in  an  earlier  chapter,  appears  to  be 
a  red  xanthophyll.  It  was  discovered  by  Monteverde  (1893)  in  the 
Russian  pond-weed,  Potamogeton  natans,  later  by  Tswett  (1911)  as 
the  cause  of  the  winter  red  color  of  the  arbor  vitse,  Thuja  orientalis, 
and  a  little  later  by  Monteverde  and  Lubimenko  (1913b)  in  the 
arillus  of  the  seed  of  the  yew,  Taxus  baccata.  The  isolation  of 
crystals  for  macroscopic  examination  can  be  carried  out  as  follows, 


METHODS  OF  ISOLATION  OF  CAROTINOIDS         217 

according  to  Monteverde  and  Lubimenko.  The  dried  material  is  first 
extracted  with  absolute  alcohol,  which  takes  out  all  the  pigments. 
The  extract  is  next  treated  with  saturated  Ba(0H)2  solution,  which 
precipitates  all  the  pigments.  The  precipitate  is  extracted  with  al- 
cohol, which  extracts  the  rhodoxanthin,  together  with  the  carotin  and 
xanthophylls,  if  present.  The  carotin  is  removed  by  shaking  with 
petroleum  ether.  This  removes  a  little  of  the  rhodoxanthin,  but  the 
bulk  of  the  pigment  remains  in  the  alcohol.  The  rhodoxanthin  shows 
great  crystallizability,  and  can  be  obtained  in  crystalline  form  merely 
by  evaporating  the  alcohol  solution,  whereas  the  xanthophyll  is  left 
as  an  amorphous  deposit.  The  rhodoxanthin  crystals  can  be  washed 
free  from  most  impurities  by  petroleum  ether,  in  which  the  pigment, 
like  xanthophyll,  is  practially  insoluble. 

Summary 

The  principles  involved  in  the  isolation  of  the  several  carotinoids 
from  plant  and  animal  tissues  are  described  in  this  chapter.  The 
methods  are  also  given  in  detail  for  the  isolation  of  crystalline  carotin 
in  quantity  from  carrots  and  green  leaves,  and  of  its  isolation  from 
animal  fat  and  blood  in  sufficient  quantity  for  macroscopic  study. 

The  evidence  is  presented  for  the  existence  of  a  carotin-albumin 
complex  in  blood  serum,  and  the  method  described  by  which  this 
can  be  isolated.  It  is  pointed  out  that  this  pigment-protein  material 
may  play  an  important  part  in  the  process  of  fat  synthesis  in  the 
mammary  gland  of  the  cow  and  that  its  further  study  may  therefore 
throw  light  on  the  formation  of  milk  fat. 

Methods  are  described  in  detail  whereby  crystalline  xanthophyll 
can  be  secured  in  quantity  from  green  leaves  and  egg  yolk,  as  well 
as  methods  for  separating  the  pigment  in  small  quantity  from  eggs 
and  blood  for  macroscopic  study. 

It  is,  pointed  out  that  xanthophyll,  in  contrast  with  carotin,  is,  in 
most  cases,  readily  extracted  from  blood  serum  by  vigorous  direct 
shaking  with  ether.  This  is  not  a  safe  basis,  however,  for  judging 
the  character  of  the  carotinoid  present  in  blood. 

The  isolation  of  crystalline  lycopin  in  quantity  from  tomatoes  is 
described,  as  well  as  the  isolation  of  fucoxanthin  from  brown  sea- 
weeds.    The  method  is  given  for  securing  crystals  of  rhodoxanthin. 


Chapter  IX 

General  Properties  and  Methods  of  Identification  of 

Carotinoids 

The  preceding  chapter  shows  clearly  the  difficulty  of  securing 
appreciable  quantities  of  the  carotinoids  in  crystalline  form.  It  is 
not  difficult,  however,  to  obtain  solutions  of  the  carotinoids  which 
show  a  number  of  characteristic  properties  that  can  serve  for  the 
identification  of  the  pigments.  For  the  sake  of  convenience,  there- 
fore, the  properties  of  the  carotinoid  solutions  and  the  properties  of 
the  crystals  of  the  pigments  will  be  presented  separately.  It  may  be 
stated  that  the  facts  to  be  presented  have  been  drawn  largely  from 
the  observations  of  Kohl,  Tswett,  Willstatter  and  his  coworkers,  to- 
gether with  the  writer's  own  experience  with  these  pigments.  These 
researches  have  already  been  referred  to  specifically  a  number  of  times 
in  the  preceding  pages. 

Properties  of  Carotinoid  Solutions 

Carotin.  Carotin  forms  well  colored  solutions  in  ether,  chloroform, 
petroleum  ether,  benzene,  carbon  tetrachloride  and  carbon  disulfide, 
as  well  as  in  ethereal  and  fatty  oils  and  oleic  acid.  The  carbon  disul- 
fide solutions  are  characterized  by  their  red  orange  to  blood  red  color. 
The  solutions  in  the  other  solvents  mentioned  are  yellow  to  golden 
yellow,  depending  on  the  concentration.  Amorphous  carotin  or  caro- 
tin in  the  presence  of  lipoids,  will  dissolve  in  95  per  cent  alcohol  or 
even  absolute  alcohol,  giving  yellow  to  golden  colored  solutions,  espe- 
cially if  hot  alcohol  is  used  for  dissolving  the  pigment.  Very  faintly 
colored  solutions  are  secured  with  dilute  alcohol,  as  a  rule.  Carotin 
crystals  are  insoluble  in  absolute  alcohol,  but  oxidation  of  carotin 
as  well  as  melting  the  crystals  greatly  increases  the  solubility  in  this 
solvent.  At  the  same  time  the  solubility  in  carbon  disulfide  decreases. 
According  to  van  den  Bergh,  Muller  and  Broekmeyer  (1920)  col- 
loidal, aqueous  solutions  of  carotin  can  be  obtained  by  a  slow  evapo- 
ration of  a  concentrated  alcoholic  solution  to  which  several  volumes 

218 


PROPERTIES  AND  METHODS  OF  IDENTIFICATION   219 

of  water  have  been  added.     This  evaporation  must  be  carried  out 
in  vacuum  aided  by  a  little  heat. 

Solutions  of  carotin  are  unaffected  by  boiling  with  alkalis,  and 
may  be  recovered  unchanged  from  such  solutions.  When  dissolved 
in  petroleum  ether  and  carbon  disulfide,  carotin  is  not  adsorbed  by 
finely  divided  substances  like  calcium  carbonate,  inulin  or  powdered 
sucrose.  However,  according  to  Tswett  (1906b),  carotin  is  adsorbed 
from  petroleum  ether  solution  by  finely  divided  HgClg,  CaClg  and 
PbS.  Miss  Stephenson  (1920)  has  reported  that  butter  fat  dissolved 
in  three  volumes  of  petroleum  ether  can  be  completely  decolorized  of 
its  carotin  by  shaking  for  several  hours  with  a  special  birchwood 
charcoal,  using  2.5  grams  per  100  grams  of  fat.  The  writer  has 
experimented  with  a  number  of  decolorizing  carbons  without  being 
able  to  duplicate  this  result.  In  strictly  adsorption  experiments  in 
which  there  was  no  indication  that  decolorization  was  due  in  part 
to  oxidation  of  the  carotin,  it  was  found  that  at  least  five  times  this 
amount  of  the  most  effective  carbon  so  far  obtainable  was  required  to 
completely  adsorb  the  carotin.  The  fact  that  carotin  is  not  adsorbed 
from  its  petroleum  ether  solution  by  calcium  carbonate  distinguishes 
the  pigment  sharply  from  some  of  the  other  carotinoids,  particularly 
the  xanthophylls.  As  a  corollary  to  this  property,  when  a  petroleum 
ether  or  carbon  disulfide  solution  of  carotin  is  filtered  through  a  column 
of  tightly  packed,  perfectly  dry  calcium  carbonate,  which  has  first  been 
moistened  with  the  solvent  (Tswett's  chromatographic  analysis)  the 
carotin  passes  through  unadsorbed.  When  carbon  disulfide  is  used  the 
zone  of  carotin  usually  has  a  characteristic  rose  color. 

Alcoholic  solutions  of  carotin  are  not  characterized  by  giving  color 
reactions  on  addition  of  concentrated  HCl,  HNO3  or  H2SO4  as  are 
certain  of  the  xanthophylls,  although  in  most  cases  the  golden  yellow 
solutions  change  slowly  to  a  deep  green  before  fading.  The  com- 
plete fading  of  this  green  solution  may  require  several  days.  The 
addition  of  NH4OH  to  the  green  solution  will  restore  the  yellow 
color,  although  the  color  is  somewhat  lighter  than  the  original,  and 
the  green  color  can  be  renewed  by  adding  acid.  Solutions  of  carotin 
in  oil  or  melted  fat  give  a  beautiful  green  color  reaction  on  dis- 
solving a  very  small  crystal  of  FegClg  in  the  warm  oil  or  fat.  A 
few  tenths  of  a  milligram  of  the  iron  salt  is  sufl&cient  to  add  to  5  cc. 
of  well  colored  oil.  The  reaction  is  very  delicate,  and  is  given  by 
xanthophylls  as  well  as  carotin.  Palmer  and  Thrun  (1916)  found 
that  this  reaction  is  caused  by  the  oxidation  of  the  carotinoid,  the 


220  CAROTINOIDS  AND  RELATED  PIGMENTS 

iron  salt  being  at  the  same  time  reduced  to  the  green  FeClg.  Huse- 
mann  (1861)  apparently  discovered  this  reaction  when  adding 
FegClg  to  an  alcoholic  solution  of  carotin,  but  it  is  doubtful  whether 
the  reaction  is  applicable  to  alcoholic  solutions  of  the  pigment  be- 
cause of  the  fact  that  alcohol  itself  will  reduce  the  red  ferric  salt  to 
the  green  ferrous  compound. 

Gill  (1917)  has  found  that  the  so-called  Crampton-Simons  test 
for  palm  oil,  in  which  a  bluish-green  color  reaction  is  given  by  an 
acetic  anhydride  reagent,  is  due  to  carotinoids  in  the  oil.  Gill's  idea 
that  carotin  alone  is  involved  is  hardly  justified,  because  the  color 
reactions  of  carotin  are  in  general  shared  by  the  other  carotinoids. 

Solutions  of  carotin  in  alcohol  which  has  been  diluted  with  water 
to  a  concentration  of  80  to  90  per  cent  alcohol  are  characterized  by 
giving  up  the  pigment  quantitatively  to  carbon  disulfide  and  petro- 
leum ether.  Conversely,  carotin  in  petroleum  ether  is  unaffected  by 
shaking  with  80  to  90  per  cent  alcohol,  even  92  per  cent  methyl  al- 
cohol failing  to  extract  any  pigment  from  the  petroleum  ether  solu- 
tion. These  properties  of  carotin,  especially  the  relatively  great  solu- 
bility in  petroleum  ether  in  comparison  with  diluted  alcohol,  serves 
to  distinguish  carotin  sharply  from  the  xanthophylls,  rhodoxanthin 
and  fucoxanthin,  and  affords  the  best  means  of  effecting  a  separation 
of  the  two  classes  of  carotinoids. 

Solutions  of  carotin  in  alcohol  and  the  fat  solvents  show  a  char- 
acteristic absorption  spectrum,  exhibiting  two,  and  under  proper  con- 
ditions three  absorption  bands  in  the  green  and  blue  part  of  the 
spectrum,  the  positions  of  the  bands  varying  somewhat  with  the  re- 
fractive index  of  the  solvent.  The  bands  are  identical  in  ether,  alco- 
hol and  petroleum  ether  because  of  the  close  agreement  in  the  indices 
of  refraction  of  these  solvents,  but  are  shifted  somewhat  towards  the 
red  in  chloroform,  which  has  a  higher  refractive  index,  and  still  fur- 
ther away  from  the  blue  in  carbon  disulfide.  The  marked  shift  of  the 
bands  into  the  brighter  part  of  the  spectrum  when  in  the  last  named 
solvent  makes  it  especially  useful  for  observing  the  spectroscopic 
properties  of  carotin,  as  well  as  the  other  carotinoids. 

Leaf  extracts  containing  chlorophyll  can  not  be  used  for  a  study 
of  the  absorption  spectra  of  the  carotinoids  because  the  absorption 
bands  of  the  chlorophylls  cover  the  second  and  third  bands  of  the 
carotinoids.  Even  the  first  carotinoid  band  coincides  very  closely 
with  Band  VIII  of  chlorophyll  b. 

The  width  and  intensity  of  the  absorption  bands  of  carotin  depend 


PROPERTIES  AND  METHODS  OF  IDENTIFICATION   221 

on  the  concentration  of  the  solution  used  and  the  depth  of  the  layer 
through  which  the  light  passes  into  the  spectroscope  and  thence  to 
the  eye  of  the  observer.  This  fact,  together  with  the  fact  that  the 
edges  of  the  absorption  bands  are  not  sharp  and  clear  cut  like  the 
lines  of  the  solar  spectrum,  no  doubt  explains  the  slight  differences 
between  the  data  given  by  various  observers  as  to  the  width  of  the 
several  absorption  bands  of  carotin  and  the  other  carotinoids.  In 
spite  of  this  fact,  however,  the  absorption  bands  of  carotin  solutions 
are  sufficiently  characteristic  to  distinguish  the  pigment  sharply  from 
the  other  carotinoids,  at  least  from  lycopin  and  the  xanthophylls  and 
rhodoxanthin.  Plate  1,  showing  a  spectrophotograph  of  the  bands 
of  carotin  and  xanthophyll  in  alcohol  and  carbon  disulfide,  brings 
out  this  point  very  clearly,  as  well  as  the  diffuse  character  of  the 
edges  of  the  bands.  It  may  be  stated,  however,  that  the  bands  may 
be  somewhat  sharper  to  the  eye  than  is  represented  in  these  photo- 
graphs. The  characteristic  feature  of  the  bands  of  carotin  which  it 
is  desired  to  point  out  is  that  in  alcohol  (an  identical  spectrum  is 
obtained  in  ether  and  petroleum  ether)  the  solar  line  F  divides  the 
first  band  almost  exactly  into  two  equal  parts.  This  is  a  character- 
istic of  the  first  carotin  band  which  may  serve  to  identify  the  caro- 
tin spectrum  from  that  of  the  other  carotinoids. 

For  direct  spectroscopic  observations  a  spectroscope  with  too  wide 
a  dispersion  may  fail  to  show  any  bands  in  a  carotin  solution  which 
exhibits  very  beautiful  bands  using  a  spectroscope  with  a  moderate 
dispersion  of  the  spectrum.  In  working  with  unknown  biological 
material  the  writer  has  had  better  success  using  an  inexpensive  spec- 
troscope with  a  moderate  dispersion  whose  spectrum  field  has  been 
standardized,  although  arbitrarily,  first  with  the  sodium  flame  and 
then  with  known  solutions  of  the  carotinoids.  Such  a  spectroscope 
set  up  in  a  dark  room  with  a  light  of  high  candle  power  concentrated 
on  the  slit  of  the  instrument  but  screened  from  the  observer,  gives 
excellent  results. 

Willstatter  and  Stoll  (1913)  give  the  following  measurements  for 
the  absorption  bands  of  carotin  in  solutions  containing  5  mg.  of  pig- 
ment per  liter,  using  a  grating  spectroscope.  These  data  correspond 
with  the  spectro-photographs  shown  in  Plate  1. 


Carotin  in  carbon 

Carotin  in  alcohol  in[i) 

disulfide  (^|x) 

6  mm.              10  m,m. 

10  mm.            20  mm. 

Band     I   

492-478            492-476 

524-510            525-508 

Band  II   

459-446           459-445 

489-475           490-474 

222  CAROTINOIDS  AND  RELATED  PIGMENTS 

Kohl  (1902b),  using  a  Zeiss  spectroscope,  obtained  the  measure- 
ments shown  in  Table  16,  using  various  solvents  with  different  re- 
fractive indices.  The  data  also  show  the  bands  of  solid  carotin, 
obtained  by  depositing  a  very  thin  layer  of  carotin  crystals  on  one 
side  of  a  glass  slide. 

Lycopin.  This  red  isomer  of  carotin  forms  yellow  solutions  in  hot 
ether,  chloroform,  alcohol,  benzene  and  petroleum  ether.  These  solu- 
tions have  a  somewhat  brown  tone  in  comparison  with  similar  solu- 
tions of  carotin.  Even  saturated  solutions  of  lycopin  in  these  sol- 
vents, with  the  possible  exception  of  chloroform,  contains  much  less 
pigment  than  the  corresponding  solutions  of  carotin.  This  probably 
accounts  for  their  yellow  color.  Solutions  of  lycopin  in  carbon  disul- 
fide are  characterized  by  their  bluish-red  color  which  persists  even  in 
great  dilution,  while  solutions  of  carotin  in  this  solvent  change  to  a 
yellowish  red  color  on  great  dilution.  The  effect  of  the  addition  of 
mineral  acids  to  alcoholic  solutions  of  lycopin  has  not  been  investi- 
gated. Lycopin,  however,  because  of  its  great  oxidizability  reacts 
toward  ferric  chloride  like  the  other  carotinoids.  The  relation  of 
lycopin  toward  adsorbents  remains  to  be  studied. 

Lycopin  shows  its  hydrocarbon  nature  by  exhibiting  the  same  rela- 
tive solubility  properties  as  carotin  when  examined  by  the  phase 
test  between  petroleum  ether  and  dilute  alcohol,  on  the  one  hand,  and 
between  dilute  alcohol  and  carbon  disulfide  on  the  other  hand.  In 
each  case  the  pigment  is  found  quantitatively  in  the  petroleum  ether 
or  carbon  disulfide. 

Table  16.    Visible  Absorption  Spectra  of  Carotin  in  Various  Solvents  with 
Different  Refractive  Index  (Kohl,  1902b) 

Refractive  Position  of  bands  {\i\i) 

Solvent  Index  Band  I  Band  II  Band  III 

Alcohol    1.358  (ave.)  490-475  455-445  430-418 

Ether 1.357  490-475  455-445  •      430-418 

Acetone  1.365  500-478  460-450  430-420 

Chloroform    1.449  505-480  465-450  435-420 

Carbon  tetrachloride   1.460  507-480  466-452  435-420 

Carbon  disulfide   1.628  510-485  470-458  437-425 

Solid  carotin    ?  550-530  495-480  460-450 

One  of  the  most  characteristic  properties  of  lycopin  solutions  which 
is  especially  serviceable  for  the  identification  of  the  pigment  is  the 
position  of  the  absorption  bands.  The  relation  of  the  lycopin  spec- 
trum in  carbon  disulfide  to  that  of  the  other  carotinoids  in  the  same 
solvent  is  shown  in  Figure  1,  taken  from  the  paper  of  Monteverde 


Spectrophotograph  of  absorption  bands  of  carotin  and 

xanthophyll    in    alcohol    and    carbon    disulfide.      (After 

Willstatter  and  Stoll) 

1.  Carotin  in  alcohol 

2.  Xanthophyll  in  alcohol 

3.  Carotin  in  carbon  disulfide 

4.  Xanthophyll  in  carbon  disulfide 


PLATE  1 


PROPERTIES  AND  METHODS  OF  IDENTIFICATION   223 


D  Eh  F 


7a 
70   65     60 


5S 


SO 


45 


4-0 


% 

^ 


f/'A 

M. 


Fig.  1.     Relative  position  of  absorption  bands  of  various  carotinoids  in 
carbon  disulfide  solution.     (After  Monteverde  and  Lubimenko) 

1.  Xanthophyll 

2.  Rhodoxanthin 

3.  Carotin  (according  to  Willstatter) 

4.  Carotin  (according  to  Monteverde) 

5.  Lycopin  (according  to  Willstatter) 

6.  Lycopin  (according  to  Monteverde) 


224  CAROTINOIDS  AND  RELATED  PIGMENTS 

and  Lubimenko  (1913b).  The  lycopin  bands  represent  the  general 
impression  which  one  obtains  when  viewing  a  solution  containing 
about  5  mg.  per  liter  at  a  depth  of  about  20  mm.  The  relative  posi- 
tions of  the  lycopin  and  carotin  bands  are  very  characteristic,  but 
they  at  once  introduce  the  difficulty  that  a  mixture  of  the  two  pig- 
ments would  show  an  almost  continuous  absorption  spectrum.  It  is 
seen,  therefore,  that  lycopin  solutions  should  be  nearly  free  from  caro- 
tin in  order  to  identify  lycopin  by  the  position  of  its  absorption  bands. 
No  means  have  yet  been  devised  for  effecting  such  a  separation  when 
the  isomers  are  present  together  in  solution.  Fractional  crystallization 
must  be  resorted  to,  and  this  is  made  possible  by  the  fact  that  lycopin 
is  much  less  soluble  than  carotin  in  almost  all  the  carotin  solvents. 
The  measurements  of  the  absorption  bands  of  lycopin  in  alcohol 
and  carbon  disulfide  are  given  by  Willstatter  and  Escher  as  follows, 
the  bands  in  carbon  disulfide  being  those  shown  by  a  standard  solu- 
tion containing  5  mg.  per  liter. 

Lycopin  in  Lycopin  in  carbon 

alcohol  (ii[i)  disulfide  (jnfx) 

10  mm.  20  mm. 

Band  I       510-499  554-540  561    -536 

Band  II     480-468  514-499.5  517.5-498 

Band  III   440-  479-472  481.5-468 

Xanthophylls.  As  pointed  out  in  Chapter  II,  the  chromatographic 
evidence  of  Tswett  seems  to  justify  the  assumption  that  several 
isomeric  xanthophylls  exist  in  nature,  in  spite  of  the  fact  that  only 
one  such  pigment  has  so  far  been  secured  in  definite  crystalline  form. 
Willstatter  has  not  yet  agreed  to  an  unqualified  acceptance  of  this 
assumption.  However,  if  the  fact  that  Tswett's  observations  can  be 
readily  verified  is  sufficient  grounds  for  accepting  his  view  of  the 
situation,  the  existence  of  more  than  one  xanthophyll  can  no  longer 
be  doubted.  At  the  same  time  it  is  recognized  that  we  owe  most  of 
our  knowledge  of  the  properties  of  xanthophyll  solutions  to  the  ob- 
servations of  Willstatter  and  Mieg  (1907),  who  first  isolated  pure 
xanthophyll  crystals  in  sufficient  quantity  to  determine  their  ele- 
mentary composition.  For  the  distinguishing  characteristics  of  the 
other  xanthophylls  which  have  not  been  crystallized  it  is  necessary 
to  refer  to  the  observations  of  Tswett  (1911). 

Xanthophylls  give  well-colored  solutions  in  a  large  number  of  sol- 
vents, including  alcohol,  ether,  acetone,  chloroform,  benzene,  carbon 
tetrachloride,  glacial  acetic  acid,  petroleum  ether,  carbon  disulfide 


PROPERTIES  AND  METHODS  OF  IDENTIFICATION   225 

and  formic  acid.  Well  colored  solutions  in  low  boiling  petroleum 
are  difficult  to  secure  because  of  the  low  solubility  of  the  pigment 
in  this  solvent,  crystalline  xanthophyll  being  almost  insoluble  in  this 
solvent.  However,  xanthophyll  in  the  amorphous  state  or  contami- 
nated with  lipoids  can  be  dissolved  quite  readily  even  in  petroleum 
ether.  The  solutions  in  all  these  solvents,  except  carbon  disulfide 
and  formic  acid,  are  yellow.  These  solutions  are  distinguished  from 
the  corresponding  carotin  solutions  by  showing  a  strong  greenish  tinge 
on  great  dilution.  The  solution  in  formic  acid,  which  is  mentioned 
only  by  Monteverde  and  Lubimenko  (1913b),  is  green.  Carotin  and 
lycopin  do  not  dissolve  in  this  solvent.  Carbon  disulfide  solutions 
of  xanthophylls  are  orange  to  orange-red,  never  blood  red  or  bluish 
red  like  carotin  and  lycopin. 

The  relative  color  intensities  of  solutions  of  carotin  and  xanthophyll 
at  equi-molar  concentrations  in  different  solvents  varies  considerably 
with  the  depth  of  the  solutions.  Willstatter  and  StoU  have  compared 
crystalline  xanthophyll  and  carotin  solutions  and  have  obtained  the 
following  results. 

5  X  10"^  MOLAR  SOLUTIONS  IN  CARBON  DISULFIDE 


Layer  oj  care 

din 

Layer  of  xantho- 

Relative 

in  mm. 

phyll  in  m,m. 

intensity 

12 

50 

1   :  4.1 

25.5 

87 

1   :  3.4 

38.5 

120 

1   :  3.1 

85 

180 

1   :  2.1 

5  X  10"^    MOL.AR    SOLUTIONS,    CAROTIN    IN    PETROLEUM    ETHEE-ETHER, 
XANTHOPHYLL   IN    ETHER 

10  20  1  :  2.0 

40  60  1   :  1.5 

91  120  1   :  1.3 

Xanthophyll  can  be  obtained  in  aqueous  colloidal  solution  in  the 
same  manner  that  colloidal  carotin  solution  is  obtained,  according 
to  van  den  Bergh,  Muller  and  Broekmeyer.  Egg  yolk  and  blood 
serum  xanthophyll  were  used  as  the  source  of  pigment  in  the  experi- 
ments by  these  investigators. 

Only  very  strong  alkali  seems  to  affect  alcoholic  solutions  of  xan- 
thophylls adversely.  Saponification  of  xanthophyll  solutions  with 
20  per  cent  alcoholic  potash  solutions  to  remove  admixed  fat,  ap- 
parently does  not  affect  the  general  properties  of  the  pigments.  Will- 
statter and  Mieg  found,  however,  that  heating  amyl  alcohol  solu- 
tions of  crystalline  xanthophyll  with  sodium  decolorized  the  pigment, 
and  heating  benzene  solutions  with  granulated  potassium  in  an  at- 


226  CAROTINOIDS  AND  RELATED  PIGMENTS 

mosphere  of  hydrogen  converted  some  of  the  pigment  into  a  product 
which  still  retained  the  solubility  of  xanthophyll  in  ether,  giving 
a  yellow  solution,  but  which  readily  formed  an  ether-insoluble  salt 
with  alkali. 

Willstatter  and  Page  (1914)  state  that  xanthophyll  is  incompletely 
recovered  by  ether  after  dissolving  in  concentrated  methyl  alcoholic 
KOH.  These  facts  all  point  to  the  possibility  of  xanthophyll  being 
attacked  by  alkalis  under  certain  conditions. 

The  effect  of  adsorbents  on  petroleum  ether  and  carbon  disulfide 
solutions  of  the  xanthophylls  is  especially  characteristic  and  serves 
not  only  to  distinguish  these  carotinoids  from  the  hydrocarbon  caro- 
tinoids  but  also  from  each  other.  Tswett  (1906b)  has  shown  that 
thoroughly  dried  precipitated  calcium  carbonate,  inulin,  sucrose  and 
many  other  compounds,  which  are  insoluble  in  petroleum  ether,  will 
completely  adsorb  the  xanthophylls  when  their  petroleum  ether  solu- 
tion is  shaken  with  an  excess  of  the  adsorbent.  In  order  to  bring 
about  this  adsorption,  however,  no  trace  of  alcohol  must  be  present, 
Tswett  having  shown  that  petroleum  ether  containing  only  one  per 
cent  alcohol  releases  the  bulk  of  the  xanthophylls  from  the  adsorbing 
agent.  There  is  therefore  good  reason  to  believe  that  much  smaller 
amounts  of  alcohol  will  interfere  greatly  with  the  adsorption. 

While  this  gross  test  may  serve  to  distinguish  the  carotinoids  con- 
taining oxygen  from  the  hydrocarbon  carotinoids,  the  principles  in- 
volved can  also  be  used  to  analyze  further  the  xanthophylls  and 
even  to  separate  them  from  each  other.  The  general  principle  which 
is  thus  utilized  is  that  when  several  substances  present  in  a  single 
solvent  are  all  adsorbed  by  a  single  adsorbent,  there  is  more  or  less 
replacement  of  one  adsorbed  substance  by  the  others,  depending  upon 
the  relative  affinities  of  the  several  substances  for  the  adsorbent,  espe- 
cially if  the  adsorption  compounds  in  each  case  are  dissociable.  This 
is  the  principle  of  Tswett's  chromatographic  analysis  of  plant  extracts 
containing  chlorophyll  and  the  carotinoids. 

The  technic  which  is  used  for  the  analysis  of  a  mixture  of  caro- 
tinoid  (and  chlorophyll)  pigments  by  this  method  is  as  follows:  A 
very  finely  divided  adsorbent  is  selected  which  will  have  no  oxidizing 
or  reducing  or  hydrolyzing  action  on  the  pigments  to  be  examined. 
Calcium  carbonate  is  especially  recommended.  Powdered  sucrose 
is  also  very  suitable.  The  calcium  carbonate  is  first  dried  for  several 
hours  at  150°  C.  A  glass  adsorption  tube  1  to  2  cm.  in  diameter  and 
10  to  15  cm.  long  is  now  prepared  which  is  drawn  out  at  one  end. 


PROPERTIES  AND  METHODS  OF  IDENTIFICATION   227 

The  small  end  is  left  with  only  a  small  opening,  1  to  2  mm.  in  diame- 
ter. A  plug  of  cotton  is  placed  in  the  small  end,  pressed  down  tightly 
and  the  tube  is  then  filled  with  the  dry  CaCOs,  which  is  poured  in 
a  little  at  a  time  and  packed  in  as  tightly  and  evenly  as  possible 
with  a  glass  rod  or  wooden  stick.  The  success  of  the  chromatograph 
depends  upon  the  evenness  with  which  the  adsorbent  is  packed  into 
the  tube.  The  tube  is  filled  within  2  or  3  cm.  of  the  top,  and  a  final 
plug  of  cotton  placed  upon  it.  The  tube  is  now  set  up  through  a 
rubber  stopper  fitted  into  a  small  filter  flask,  gentle  suction  applied 
and  a  stream  of  pure  solvent  (either  petroleum  ether  or  carbon  disul- 
fide, depending  on  the  solvent  selected  for  the  pigment  solution) 
passed  through  the  column  until  the  adsorbent  is  moistened  with 
it.  The  suction  is  stopped  and  the  upper  cotton  plug  removed.  Suf- 
ficient pigment  solution  is  now  poured  into  the  tube  to  color  about 
1  cm.  of  the  adsorbent.  When  this  has  passed  into  the  column  with 
the  aid  of  gentle  suction,  the  tube  is  filled  with  solvent  and  suction 
continued.  The  upper  part  of  the  tube  is  kept  filled  with  pure  solvent 
in  order  to  establish  a  stream  of  the  solvent  through  the  adsorbing 
colunm.  The  layer  of  pigment  will  now  pass  through  slowly  and  will 
differentiate  itself  into  zones  of  relative  adsorption,  thab  of  greatest 
adsorption  affinity  being  at  the  top,  and  that  of  the  least  at  the  bottom. 
Inasmuch  as  all  the  chlorophylls  and  carotinoids  form  dissociable  com- 
pounds with  CaCOs  the  stream  of  pure  solvent  will  slowly  wash  them 
through  the  column  as  differently  colored  zones.  If  the  column  has 
been  packed  perfectly  evenly  with  the  adsorbent  the  zones  will  be 
true  rings,  otherwise  they  will  be  irregular.  Perfectly  true  adsorp- 
tion rings  are  difficult  to  secure.  Pigments  obtained  in  the  various 
zones  by  this  method  are  not  pure,  as  Tswett  has  pointed  out,  but 
can  be  purified  by  repeating  the  analysis  on  the  pigment  obtained 
in  any  desired  zone. 

A  chromatographic  analysis  applied  in  the  above  manner  to  a  pe- 
troleum ether  or  carbon  disulfide  solution  of  carotin  and  the  four 
xanthophylls  recognized  by  Tswett  should  show  the  following  result. 
Assuming  that  carbon  disulfide  has  been  used  and  the  differentiation 
has  been  continued  with  a  stream  of  solvent  until  the  least  adsorbed 
pigment  has  reached  the  bottom  of  the  column  the  lowest  zone  will  be 
rose  colored  due  to  carotin;  above  this,  probably  separated  by  a 
more  or  less  colorless  region,  will  be  a  wide  orange-yellow  zone,  due  to 
xanthophyll  a,  which  apparently  comprises  the  major  part  of  the 
xanthophylls;    still  higher   in  the   column   and   separated   from   the 


228  CAROTINOIDS  AND  RELATED  PIGMENTS 

xanthophyll  a  will  be  a  yellow  zone  due  to  xanthophylls  a'  and 
a'',  whose  differentiation  will  be  described  in  a  moment;  near  the  top 
of  the  column  will  be  a  narrow  yellow  zone,  due  to  xanthophyll  (3. 
If  a  stream  of  benzene  is  run  through  the  column  at  this  point,  the 
carotin  and  xanthophyll  a  will  be  quickly  washed  away  and  the  yellow 
zone  containing  xanthophylls  a'  and  a^  will  slowly  separate  into  two 
zones.  These  can  now  be  washed  out  of  the  column  with  petroleum 
ether  containing  one  per  cent  absolute  alcohol,  leaving  xanthophyll  (3 
still  adsorbed.  This  pigment  can  be  removed,  however,  by  petro- 
leum ether  containing  10  per  cent  absolute  alcohol. 

While  a  chromatographic  analysis  of  an  unknown  pigment  solution 
is  instructive  it  does  not  necessarily  provide  a  means  of  definite  iden- 
tification of  any  xanthophyll  pigments  which  may  be  present.  Any 
pigments  differentiated  by  this  test  must  be  submitted  to  further  ex- 
amination. When  several  pigments  are  shown  by  such  an  analysis,  a 
second  chromatographic  separation  should  be  carried  out  on  a  solution 
of  each  of  the  pigments  for  the  purpose  of  purifying  it  as  far  as  pos- 
sible. Comparison  can  then  be  made  with  the  known  properties  of 
solutions  of  xanthophylls  a,  a,  a!'  and  /3,  which  are  as  follows: 

Xanthophyll  a.  This  pigment  is  quantitatively  removed  by  80-90 
per  cent  alcohol,  preferably  methyl  alcohol,  from  its  solution  in  petro- 
leum ether.  It  is  adsorbed  by  an  excess  of  CaCOg  from  pure,  abso- 
lutely alcohol-free,  low  boiling  petroleum  ether.  It  is  the  least  ad- 
sorbed by  CaCOs  from  CSg  of  any  of  the  xanthophylls.  Its  carbon 
disulfide  solutions  are  orange  to  red  orange.  Its  alcoholic  solution  is 
bleached  by  addition  of  concentrated  mineral  acids,  passing  through 
a  green  color  before  fading.  Its  spectroscopic  absorption  bands  are 
identical  with  those  of  crystalline  xanthophyll.  Plate  1  shows  these 
bands  in  alcohol  and  carbon  disulfide  in  comparison  with  those  of 
carotin.  The  measurements  of  these  bands,  using  a  solution  contain- 
ing 5  mg.  of  pigment  per  liter,  are  stated  by  Willstatter  and  Stoll 
to  be  as  follows: 


Xanthophyll  in 
alcohol  (|xn) 

Xanthophyll  in  carbon 
disulfide  ([i[i) 

5  mm.            10  mm. 

10  mm.            20  mm. 

Band  I       

484-472           488-471 

515-501           516-501 

Band  II     

454rA'il            454-440 

482-469            483-467 

Band  III    

41&-                 420- 

447-441 

Xanthophylls  a    and  a'.    These  pigments  are  quantitatively  ex- 
tracted from  petroleum  ether  by  80-90  per  cent  alcohol,  preferably 


PROPERTIES  AND  METHODS  OP  IDENTIFICATION    229 

methyl  alcohol.  They  are  readily  adsorbed  from  petroleum  ether  by 
CaCOg  and  more  readily  adsorbed  from  carbon  disulfide  by  CaCOg 
than  xanthophyll  a.  The  carbon  disulfide  solutions  are  yellow  to 
orange.  The  pigments  are  readily  released  from  adsorption  on  CaCOg 
by  benzene  and  when  thus  adsorbed  in  a  chromatogram  may  be 
separated  from  each  other  by  this  solvent.  The  action  of  concen- 
trated mineral  acids  from  the  alcoholic  solution  of  these  xanthophylls 
is  not  known,  but  it  may  be  similar  to  that  on  xanthophyll  p.  The 
absorption  bands  of  these  xanthophylls  is  stated  by  Tswett  to  be 
shifted  slightly  towards  the  violet  from  those  of  xanthophyll  a.  The 
measurements  of  these  bands  has  not  been  reported. 

Xanthophyll  /?.  This  carotinoid,  like  the  other  xanthophylls,  is 
quantitatively  extracted  from  petroleum  ether  by  80-90  per  cent  alco- 
hol. It  forms  almost  undissociable  adsorption  compounds  with  CaCOg 
when  in  petroleum  ether  or  carbon  disulfide,  but  can  be  released  from 
this  combination  by  petroleum  ether  containing  10  per  cent  absolute 
alcohol.  Concentrated  mineral  acids  produce  a  green  color,  passing 
to  a  peacock  blue  when  added  to  its  alcoholic  solution.  NH^OH  will 
restore  the  yellow  color  and  acid  the  blue  color.  The  reaction  is  similar 
to  one  shown  by  fucoxanthin,  in  which  a  hydrochloride  is  formed,  and 
in  which  the  yellow  pigment  restored  by  alkali  still  retains  one  mole- 
cule of  HCl.  The  absorption  bands  of  alcoholic  solutions  of  xan- 
thophyll p  lie  at  475-462[i[x  and  445-43 l^ijx,  which  are  seen  to  be 
shifted  appreciably  towards  the  violet  from  the  bands  of  crystalline 
xanthophyll. 

Rhodoxanthin.  This  red  isomer  of  the  xanthophylls  is  known 
largely  through  the  properties  of  its  solutions,  pure  crystals  of  the 
pigment  not  yet  having  been  obtained  in  suflBcient  quantity  for 
analysis.  This  carotinoid  forms  yellow  solutions  in  petroleum  ether, 
ether  and  benzene,  like  other  carotinoids,  but  its  alcoholic  and  acetone 
solutions  are  rose  colored  or  pink.  It  is  also  dissolved  by  glacial 
acetic  acid  with  a  red  color.  The  red  color  in  certain  solvents  serves 
to  distinguish  the  pigment  from  other  carotinoids,  as  does  also  the 
ruby  red  or  violet  red  color  in  carbon  disulfide.  Formic  acid  also 
dissolves  the  pigment,  at  first  with  a  pink  color  which  later  turns 
yellow. 

Rhodoxanthin,  in  common  with  other  carotinoids,  is  not  readily  at- 
tacked by  alkali.  Its  xanthophyll-like  character  is  shown  by  the  fact 
that  80  per  cent  alcohol  quantitatively  extracts  the  pigment  from  its 
solution  in  petroleum  ether.    In  common  with  crystalline  xanthophyll 


230  CAROTINOIDS  AND  RELATED  PIGMENTS 

the  crystals  show  only  very  slight  solubility  in  petroleum  ether.  When 
its  petroleum  ether  or  carbon  disulfide  solutions  are  analyzed  by  means 
of  the  chromatograph  the  pigment  shows  very  little  adsorption  affinity 
for  CaCOg,  its  adsorption  zone  preceding  all  the  others  in  a  chromato- 
graphic analysis  of  extracts  obtained  from  leaves  in  which  the  pig- 
ment abounds,  like  the  winter  foliage  of  arbor  vitse  {Thuja  orientalis). 
When  carbon  disulfide  is  employed  as  solvent  the  rhodoxanthin  zone 
has  a  characteristic  ruby  red  color. 

Solutions  of  rhodoxanthin  show  three  absorption  bands  in  a  char- 
acteristic position  in  the  spectrum,  being  shifted  farther  towards  the 
red  than  any  of  the  other  carotinoids.  The  position  of  the  bands, 
taken  from  the  observations  of  Monteverde  and  Lubimenko  (1913b), 
which  appear  to  be  the  most  accurate,  are  as  follows: 

Inpetroleum  In  carbon 

ether  (ji[x)  disulfide  {[xii) 

Band  I       530-513  575-553 

Band  II     495-480  535-515 

Band  III    470-455  500-480 

The  relation  of  these  bands,  when  in  carbon  disulfide,  to  the  bands 
of  the  other  carotinoids  in  the  same  solvent  is  shown  in  Figure  1. 

The  effect  of  mineral  acids  upon  the  alcoholic  solution  of  rhodoxan- 
thin has  apparently  not  been  determined. 

Fucoxanthin.  This  carotinoid,  which  is  characteristic  of  the  brown 
algse,  gives  well  colored  solutions  in  practically  all  the  organic  solvents. 
Although  the  pure  crystals  are  completely  insoluble  in  petroleum 
ether,  the  presence  of  lipoids  makes  it  possible  to  obtain  colored  solu- 
tions in  this  solvent  also.  This  is  likewise  true  of  methyl  alcohol  in 
which  the  pure  crystals  are  very  sparingly  soluble.  The  ether  solu- 
tion of  fucoxanthin  is  orange  yellow,  the  alcoholic  solutions  have  a 
somewhat  rusty,  or  brownish  yellow  tinge,  and  the  carbon  disulfide 
solution  is  deep  red.  Fucoxanthin  is  a  more  intense  pigment  than 
either  carotin  or  crystalline  xanthophyll.  Willstatter  and  Page  (1914) 
have  stated  that  a  comparison  of  5  x  10~^  molar  solutions  of  the  three 
pigments  in  ether  shows  that  50  mm.  of  fucoxanthin  is  equal  in  color 
to  80  mm.  of  the  carotin  and  108  mm.  of  xanthophyll. 

The  effect  of  adsorbents  on  petroleum  ether  and  carbon  disulfide 
solutions  of  fucoxanthin  has  not  been  studied,  but  the  very  low  solu- 
bility of  the  pigment  in  petroleum  ether  suggests  that  it  would  be 
readily  adsorbed  from  this  solvent  by  CaCOg. 

Solutions  of  fucoxanthin  show  two  well  defined  absorption  bands, 


PROPERTIES  AND  METHODS  OF  IDENTIFICATION    231 

but  the  positions  of  the  bands  are  not  sufficiently  characteristic  to 
distinguish  the  pigment  sharply  from  carotin  or  xanthophyll.  The 
bands  of  the  alcoholic  solution,  containing  5  mg.  per  liter  are  given 
by  Willstatter  and  his  co-workers  as  follows: 

Fucoxanthin  in  alcohol  {]x\x) 
5  mm.  layer  10  mm.layer   20  mm.  layer 

Band  I     486-469  492-476  498-473 

Band  II   455-440  467-451  462-443 

End  Absorption  440-. . .  

One  of  the  most  characteristic  properties  of  fucoxanthin  solutions 
which  can  be  used  as  an  aid  in  identification  as  well  as  a  means  of 
separation  of  the  pigment  from  other  carotinoids  is  the  fact  that  70 
per  cent  methyl  alcohol  will  quantitatively  extract  the  pigment  from 
its  solution  in  petroleum  ether — ethyl  ether  (1:1).  This  fact  has  al- 
ready been  pointed  out  in  connection  with  the  isolation  of  the  caroti- 
noids, and  is  especially  useful  in  the  quantitative  estimation  of  fuco- 
xanthin as  will  be  shown  in  the  next  chapter. 

Fucoxanthin  solutions  are  very  much  less  stable  than  those  of  the 
other  carotinoids,  particularly  in  the  light.  Benzene  solutions  bleach 
especially  readily.  Ether  solutions  of  fucoxanthin  give  a  reaction 
with  HCl  which  resembles  in  many  respects  the  action  of  mineral 
acids  on  alcoholic  solutions  of  xanthophyll  (3.  When  the  ether  solu- 
tion of  pigment  is  shaken  with  30  per  cent  HCl  solution  the  pig- 
ment bleaches  and  the  acid  layer  takes  on  a  magnificent  blue-violet 
or  sky-blue  color.  The  latter  is  due  to  a  stable  salt  containing  4 
atoms  of  HCl,  which  is  probably  an  oxonium  compound.  Its  solu- 
bility in  the  aqueous  layer  is  due  only  to  the  ether  which  is  dis- 
solved in  the  acid  solution.  On  regeneration  of  the  yellow  pigment 
with  alkali,  the  hydrochloride  still  persists  and  retains  one  atom  of 
HCl.  Fucoxanthin  apparently  unites  with  other  substances  as  well 
as  HCl  for  Willstatter  and  Stoll  state  that  ether  solutions  dried  over 
CaClg  yield  a  pigment  showing  3  to  4  per  cent  CaO. 

Another  especially  characteristic  property  of  fucoxanthin  is  the 
action  of  alkalis  on  its  solutions,  or  rather  on  the  pigment  itself  when 
in  solution.  The  pigment  apparently  has  no  acid  properties  but 
under  certain  conditions  it  is  attacked  by  alkali.  Metallic  sodium, 
solid  Ba(0H)2  and  50  per  cent  KOH  have  no  effect  upon  it.  It  is 
dissolved,  however,  by  strong  aqueous  KOH  solutions,  and  cannot 
be  extracted  from  this  solution  by  ether.    This  is  also  true  of  con. 


232  CAROTINOIDS  AND  RELATED  PIGMENTS 

methyl  alcohol  KOH,  and  in  this  solvent  the  pigment  is  changed  so 
that  on  dilution  with  water  and  extraction  with  ether  (the  pigment 
being  liberated  from  its  temporary  alkali  compound  by  water)  it  is 
much  more  sensitive  towards  HCl.  Ether  solutions  will  now  give 
the  blue  color  reaction  on  shaking  with  only  16  per  cent  HCl  solu- 
tion, whereas  25  per  cent  HCl  solutions  had  scarcely  any  effect  before 
the  treatment  with  alkali.  Even  3  per  cent  HCl  now  has  a  noticeable 
effect,  and  in  concentrated  ether  solution  even  0.001  per  cent  HCl 
will  give  the  blue  color.  The  hydrochlorides  formed  in  these  cases 
apparently  contain  even  more  chlorine  than  the  hydrochloride  which 
the  original  pigment  forms. 

The  ether  solution  of  fucoxanthin  which  has  been  changed  by  the 
concentrated  methyl  alcohol  KOH  has  a  greenish  tinge  and  shows  a 
spectrum  whose  bands  are  shifted  considerably  towards  the  violet. 
The  ether  solution  of  this  pigment  containing  5  mg.  per  liter  shows 
bands  at  461-451|X[x  and  435-423n[x  in  10  mm.  layer. 

Properties  of  Crystalline  Carotinoids 

Carotin.  Carotin  crystallizes  from  carbon  disulfide  on  addition  of 
absolute  alcohol,  forming  rhombic  tablets  or  prisms,  and  from  petro- 
leum ether,  forming  almost  quadratic  leaflets,  which  are  frequently 
indented.  Plate  2,  figure  1,  shows  the  form  of  crystals  from  carbon 
disulfide-alcohol.  The  color  of  the  crystals  varies  with  their  thick- 
ness from  bright  yellow  to  deep  rose  or  copper  colored  with  a  rich 
velvety  appearance.  The  crystals  are  highly  pleochromatic  and  have 
an  intense  blue  to  bright  green  metallic  luster  by  reflected  light.  The 
crystallography  of  carotin  has  been  described  in  detail  by  Kohl 
(1902b).  Some  investigators  have  ascribed  a  striking  violet  or  crocus- 
like odor  to  the  pure  crystals,  but  this  has  not  been  observed  by 
others,  e.g.,  Willstatter.  The  crystals  from  alcohol  usually  contain 
some  alcohol  of  crystallization,  which  is  given  up  in  vacuum  over 
H2SO4  or  P2O5. 

Pure  carotin  crystals  are  almost  insoluble  in  cold  ethyl  alcohol, 
and  even  less  so  in  methyl  alcohol.  They  dissolve  with  difficulty  in 
the  hot  alcohols.  About  1.5  liters  of  low  boiling  petroleum  ether  are 
required  to  dissolve  one  gram,  under  a  reflux  condenser,  but  the  solu- 
bility is  somewhat  greater  in  the  higher  boiling  gasoline  fractions. 
About  900  cc.  of  hot  ether  are  required  for  one  gram  of  crystals. 
Acetone  dissolves  the  crystals  with  difficulty,  even  hot  acetone  not 


41 


\   , 


pr 


..  1^.    #*     „\ 


9^   *  ^      •  / 


^^    * 


Fig.  1.    Carotin  from  carbon  disulfide-       Fig.  2.    Xanthophyll  from  methyl  al- 
alcohol.     (X62)  cohol.     (X62) 


Fig.  3.    Xanthophyll  iodide  from 
alcohol. 


Fig.  5.     Lycopin  from  carbon  disul- 
fide-alcohol.     (X165) 


Fig.  4.     Lycopin  from  petroleum 
ether.     (X  165) 


Fig.  6.    Fucoxanthin  from  methyl 
alcohol. 


PLATE  2 


PROPERTIES  AND  METHODS  OF  IDENTIFICATION    233 

being  a  ready  solvent.  Benzene  dissolves  the  pure  crystals  much  more 
easily  and  chloroform  and  carbon  disulfide  with  great  ease.  Kohl 
(1902e)  gives  the  specific  rotation  of  carotin  in  chloroform  as 
a  D  =  —30.17°,  but  this  property  is  not  mentioned  by  other  investi- 
gators. 

Carotin  crystals,  and  even  the  amorphous  pigment,  if  free  from 
lipoids,  dissolve  in  concentrated  H2SO  with  an  indigo  blue  color,  from 
which  the  pigment  precipitates  as  green  flakes  on  dilution,  A  similar 
color  reaction  is  given  by  concentrated  HNO3,  dry  sulphurous  acid 
and  by  thymol  and  phenol  containing  concentrated  HCl.  The  crys- 
tals also  give  a  transient  blue  color  with  bromine  water  and  with 
bromine  vapor.  With  ferric  chloride  a  deep  green  color  is  given. 
This  reaction  was  explained  in  a  previous  paragraph.  The  color 
reaction  of  carotin  with  H2SO4,  which  is  also  given  by  the  other 
carotinoids,  is  regarded  by  many  as  a  specific  reaction  for  these 
pigments.  There  is  no  justification  for  this  idea,  which  may  easily 
lead  to  erroneous  conclusions,  because  this  reaction  is  given  by  a 
large  number  of  organic  compounds,  especially  by  certain  quinones  of 
the  aromatic  group. 

The  crystals  of  carotin  readily  oxidize,  whereby  the  crystals  bleach 
entirely.  The  original  melting  point  of  167.5°-168°  C.  falls  and  the 
pigment  changes  markedly  in  properties.  A  number  of  investigators 
have  reported  that  the  bleached  pigment  shows  the  color  reactions 
of  cholesterol,  but  Willstiitter  and  Mieg  and  Euler  and  Nordenson  were 
unable  to  confirm  this.  The  amount  of  oxygen  which  carotin  is  ca- 
pable of  taking  up  during  the  oxidation  has  been  variously  reported, 
Arnaud  reporting  21  to  24  per  cent,  Kohl  as  high  as  37.87  per  cent. 
Willstatter  and  Mieg  obtained  a  maximum  of  34.3  per  cent,  cor- 
responding to  11  atoms  of  oxygen.  Willstatter  and  Escher  (1910)  ob- 
tained an  oxidized  product  in  dry  oxygen  corresponding  to  nearly  12 
atoms  of  oxygen.  They  found  that  oxidation  in  a  room  saturated  with 
moisture  gave  a  product  with  a  like  amount  of  oxygen  but  containing 
2  molecules  of  water,  in  addition.  The  perfectly  pure  pigment  crys- 
tals did  not  oxidize  readily.  By  placing  them  in  a  stream  of  pure 
oxygen  the  increase  in  weight  was  only  0.3  per  cent  after  five  days. 
After  this  the  oxidation  was  more  rapid,  and  was  accompanied  by  the 
violet-like  odor  which  had  been  described  by  others  for  the  pure  pig- 
ment. 

Carotin  being  an  unsaturated  hydrocarbon  would  be  expected  to  form 
stable  halogen  derivatives.    Two  iodides  have  been  described,  one  by 


234  CAROTINOIDS  AND  RELATED  PIGMENTS 

Arnaud  (1886)  which  would  correspond  to  the  formula  C4oH5el3,  ac- 
cording to  our  present  accepted  composition  of  the  hydrocarbon.  This 
iodide  is  formed  by  adding  iodine  to  a  petroleum  ether  solution  of  caro- 
tin crystals  in  less  than  the  required  amount  to  combine  with  all  the 
carotin.  The  iodide  crystallizes  out  as  dark  violet  leaflets  with  a  cop- 
per colored  reflection,  which  melt  sharply  at  136°-137°  C.  Willstat- 
ter  and  Escher  obtained  the  same  iodide  by  adding  double  the  amount 
of  iodine  crystals  to  benzene-carbon  disulfide  and  carbon  disulfide- 
ether  solutions  of  carotin.  The  other  iodide,  described  by  Willstatter 
and  Mieg  and  also  by  Willstatter  and  Escher  (1910),  corresponds  to 
the  formula  C^oHgglg,  and  is  prepared  by  adding  crystalline  iodine  to 
an  ether  solution  of  carotin  in  an  amount  equal  to  only  one-third  the 
weight  of  carotin  present.  The  form  and  color  of  these  crystals  are 
the  same  as  those  of  the  tri-iodide,  but  differ  from  it  by  showing  no 
definite  melting  point,  the  crystals  slowly  decomposing  between  140° 
and  170°  C. 

The  analyses  which  have  been  made  of  the  two  iodides,  one  contain- 
ing two  atoms  and  the  other  three  atoms  of  the  halogen,  show  ex- 
cellent correspondence  with  the  theoretical  amount  of  iodine  in  com- 
pounds showing  this  composition.  It  is  not  clear,  however,  just  what 
structure  of  the  carotin  molecule  would  permit  the  formation  of  an 
iodide  containing  three  atoms  of  iodine. 

Carotin  also  forms  a  bromine  derivative  which  is  at  once  both  an  ad- 
dition and  a  substitution  product,  which  conforms  with  the  constitution 
C^qH-soBtzz-  The  formation  of  this  product  is  stated  by  Willstatter 
and  Escher  to  take  place  on  the  addition  of  0.5  grams  of  powdered 
carotin  in  small  portions  with  shaking  to  16  grams  of  bromine  at  0°  C, 
the  bromine  being  protected  from  moisture  in  the  reaction  flask  by  a 
CaClg  tube.  The  reaction  is  completed  by  standing  at  room  tempera- 
ture and  the  precipitate  filtered  off  on  glass  wool  and  washed  with  hot 
anhydrous  formic  acid,  giving  a  brittle  colorless  product  without  defi- 
nite crystalline  form.  The  bromide  has  no  melting  point  but  de- 
composes at  about  171°— 174°  C.  It  dissolves  easily  in  benzene  and 
carbon  disulfide,  fairly  easily  in  ether,  but  with  difficulty  in  even  hot 
alcohol  or  petroleum  ether;  it  is  insoluble  in  glacial  acetic  and  an- 
hydrous formic  acid.  The  bromine  cannot  be  completely  removed  with 
zinc  dust  and  glacial  acetic  acid,  or  with  silver  acetate. 

The  structure  of  the  carotin  molecule  has  proved  to  be  a  difficult 
problem  to  solve.  Escher's  (1910)  attempt  resulted  only  in  the  pro- 
duction of  amorphous  products  of  high  molecular  weight.    One  nat- 


PROPERTIES  AND  METHODS  OF  IDENTIFICATION    235 

urally  wonders  whether  any  of  the  known  compounds  of  the  same 
empirical  formula  bear  any  relation-to  carotin,  but  it  must  be  admitted 
that  they  throw  no  light  on  its  constitution.  The  simplest  empirical 
formula  for  carotin,  i.e.,  (C5H7)8  suggests  a  possible  relation  to  the 
terpene  derivatives,  the  cymenes,  or  to  the  toluene  derivatives,  durene 
and  the  propyl  toluenes,  all  of  which  have  the  empirical  formula 
C10H14.  The  cymenes,  however,  are  structurally  isopropyl  benzenes, 
and  durene  is  tetramethyl  benzene.  The  former  is  a  colorless  oil  and 
the  latter  a  colorless  solid,  m.p.,  79-80°  C.  There  seems  to  be  no  rea- 
son for  believing  that  carotin  is  related  in  any  way  to  these  substances 
or  to  the  propyl  toluenes. 

The  problem  of  the  structure  of  carotin  is  of  special  interest  because 
pigmented  hydrocarbons  are  something  of  a  novelty.  Those  which 
have  been  described  will  be  mentioned  briefly,  inasmuch  as  their  con- 
stitution at  least  indicates  the  probably  chromatophor  group  in  caro- 
tin, namely,  >C:C<. 

Apparently  the  first  pigmented  hydrocarbon  to  be  mentioned  in  the 
literature   is   acenaphtylene,   CiaHg,   probably   having  the   structure 

1.    It  was  first  described  by  Behr  and  van  Dorp  (1873)  and 


HC:CH 

Blumenthal  (1874)  and  later  by  Graebe  (1893).  It  forms  leaflets 
of  a  golden  yellow  color,  soluble  in  alcohol,  ether  and  benzene,  which 
melt  at  92°-93°  C.  De  la  Harpe  and  van  Dorp  (1875)  and  later 
Graebe  (1892)  have  described  the  hydrocarbon  di-biphenylenathene, 
CgeHie,  which  crystallizes  as  intensely  yellowish  red  needles  and 
scales,  m.p.,  187°-188°  C.  (corrected),  and  forming  yellow  solutions. 
According  to  Graebe  this  hydrocarbon  adds  hydrogen  readily,  going 
over  into  the  colorless  compound  CgeHig.  Thiele  (1900a)  found  that 
the  hydrocarbon  fulven,  CaH^.CiC.CHa  forms  brilliantly  colored  de- 
rivatives. He  mentions  dimethyl  fulvene,  CgHio,  a  bright  orange  col- 
ored oil,  methyl  phenyl  fulvene,  CigHig,  a  red  oil,  and  diphenyl  ful- 
vene, CigHi^,  which  crystallizes  from  petroleum  ether  as  deep  red 
prisms,  m.p.,  82°  C.  Thiele  (1900b)  has  also  described  the  hydro- 
carbon cinnamylidenindene,  CsHg.CHiCH.C  —  CHiCII,  which  crys- 

\        / 

tallizes  as  yellowish-red  needles,  m.p.,  190°  C.,  and  is  easily  soluble 
in  most  organic  solvents.     More  recently  Pummerer  (1912)  has  dis- 


236  CAROTINOIDS  AND  RELATED  PIGMENTS 

covered  the  red  hydrocarbon,  CzJi^^,  m.p.,  306°  C,  which  he  calls 
rubicen,  and  which  dissolves  in  CHCI3,  giving  solutions  showing  an 
intense  yellow  fluorescence.  It  is  not  readily  soluble  in  the  other  or- 
ganic solvents.  The  chloroform  solution  shows  one  absorption  band 
with  a  maximum  at  498[.i[x.  The  spectroscopic  properties  of  the  other 
colored  hydrocarbons  mentioned  has  apparently  not  been  determined. 

Apparently  color  among  hydrocarbons,  although  rare,  is  not  con- 
fined to  yellow  and  red.  Sherndal  (1915)  has  isolated  a  blue  hydro- 
carbon oil,  azulene,  C15H18,  from  a  number  of  essential  oils.  It  is  a 
coincidence,  perhaps  worth  mentioning,  that  this  blue  hydrocarbon 
dissolves  in  60  to  65  per  cent  sulfuric  acid  with  a  yellow  color, 
whereas  carotin,  a  red  hydrocarbon,  dissolves  in  strong  sulfuric  acid 
with  a  blue  color. 

In  addition  to  these  substances  Marchlewski  (1903)  called  atten- 
tion to  the  fact  that  pigmented  compounds  could  be  made  start- 
ing with  methyl-ethyl  maleic  acid  anhydride,  which  show  a  strong 
resemblance  to  the  lipochromes  both  with  respect  to  spectroscopic 
properties  and  color  reactions.  Marchlewski's  note  on  the  subject 
was  for  the  purpose  of  reserving  the  field  of  investigation,  but  so 
far  as  the  writer  is  aware  no  further  results  have  ever  been  published. 

Xanthophyll.  The  crystals  of  xanthophyll  obtained  from  plants 
by  Willstatter  and  Mieg  (1907)  and  from  egg  yolk  by  Willstatter 
and  Escher  show  complete  correspondence  in  form,  color,  solubility, 
oxidation  products  and  halogen  derivatives,  but  not  in  melting  point. 
The  latter  point  was  discussed  in  Chapter  VI. 

Xanthophyll  appears  to  crystallize  best  from  alcohol,  preferably 
methyl  alcohol,  from  which  the  forms  are  mostly  quadratic,  often 
trapesium  tablets,  frequently  showing  indentations.  Their  general 
appearance  is  shown  in  Plate  2,  Figure  2.  From  ethyl  alcohol  the 
crystals  are  lance-  and  wedge-shaped  prisms.  Sometimes  the  crystals 
are  rhombic,  almost  hexahedrons.  In  all  cases  the  crystals  contain  a 
molecule  of  the  solvent  from  which  crystallization  occurred.  A  sin- 
gle example  is  reported  by  Willstatter  and  Mieg  in  which  crystals 
from  CSg  contained  22  per  cent  sulphur. 

The  color  of  the  crystals  varies  with  the  thickness  from  a  green- 
ish-yellow to  a  rose,  similar  to  the  crystals  of  carotin  but  distin- 
guished by  a  less  red  color.  The  crystals  are  even  more  strongly 
pleochromatic  than  carotin,  their  brilliant  steel  blue  reflection  being' 
especially  evident  when  suspended  in  the  solvent.  The  powdered 
crystals  have  a  brick  red  to  red-lead  color.    After  removing  the  sol- 


PROPERTIES  AND  METHODS  OF  IDENTIFICATION   237 

vent  of  crystallization  by  drying  in  vacuum  over  H2SO4  or  PgOg  xan- 
thophyil  crystals  from  plants  melt  at  173.5°-174.5°  C.  (corrected), 
but  according  to  Willstatter  and  Escher  xanthophyll  from  egg  yolk 
melts  at  195°-196°  C.  (corrected). 

Xanthophyll  crystals  are  entirely  insoluble  in  low  boiling  petro- 
leum ether.  The  solubility  in  cold  methyl  alcohol  is  quite  low,  1  gram 
requiring  about  5  liters,  but  is  considerably  greater  in  the  boiling  sol- 
vent, 1  gram  requiring  700  cc.  to  1  liter.  The  solubility  of  the  pure 
pigment  in  ethyl  alcohol  is  considerably  greater.  The  crystals  dis- 
solve rather  easily  in  warm  CSg,  1  gram  requiring  about  400  cc.  of 
solvent.  The  solubility  in  ether  is  a  little  greater  and  in  acetone  and 
chloroform  quite  rapid.  Phenol  also  dissolves  the  crystals  quickly 
as  does  hot  glacial  acetic  acid.  A  mixture  of  3  parts  phenol  crystals 
and  1  part  glycerol  also  dissolve  xanthophyll  crystals  very  readily, 
as  van  Wisselingh  (1915)  has  shown. 

Xanthophyll  crystals,  like  carotin,  dissolve  in  con.  H2SO4.  with  a 
deep  blue  color,  from  which  green  flakes  are  precipitated  on  dilution 
with  water.  They  also  dissolve  in  warm  ethyl  alcohol  containing 
strong  HCl  with  a  pure  green  color  which  soon  changes  to  blue.  This 
reaction  is  apparently  peculiar  to  xanthophyll  in  contrast  with  the 
hydrocarbon  carotinoids.  According  to  van  Wisselingh  xanthophyll 
crystals  can  be  distinguished  from  carotin  crystals  by  the  fact  that 
the  former  are  colored  blue  but  are  not  dissolved  by  65  to  75  per  cent 
H2SO4  while  the  latter  turn  blue  only  after  some  lapse  of  time  or  when 
stronger  acid  is  used.  With  con.  HNO3  a  colorless  solution  only  is 
obtained  from  which  colorless  flakes  separate.  This  is  Willstatter 
and  Mieg's  finding,  but  van  Wisselingh  found  that  the  blue  color  re- 
action resulted  with  50  per  cent  acid.  This  may  also  serve  to  dis- 
tinguish the  crystals  of  the  two  types  of  carotinoids. 

Xanthophyll,  like  carotin,  is  unsaturated  and  forms  an  iodide, 
C4oH5g02l2,  which  precipitates  at  once  on  addition  of  iodine  to  the 
ethereal  solution  of  the  pigment.  An  excess  of  iodine  prevents  the 
crystallization.  The  iodide  has  a  dark  violet  color  and  consists  of 
tuft-like  prisms,  the  form  of  which  is  shown  in  Plate  2,  Figure  3.  The 
compound  is  not  very  stable  and  possesses  no  definite  melting  point. 
It  is  fairly  readily  soluble  in  the  xanthophyll  solvents,  excepting 
ether,  giving  yellow  to  yellowish-red  solutions.  When  xanthophyll 
is  brominated  it  loses  its  oxygen,  since  Willstatter  and  Escher  (1910) 
report  a  xanthophyll  bromide  with  the  constitution  C4oH4oBr22. 

Xanthophyll  crystals  slowly  oxidize  in  the  air  or  in  oxygen,  with 


238  CAROTINOIDS  AND  RELATED  PIGMENTS 

the  addition  of  36.5  per  cent  of  their  original  weight,  corresponding 
to  13  atoms  of  oxygen  or  the  formula  C4oH5eOi5.  When  this  product  is 
recrystallized  from  ether  it  contains  even  more  oxygen  and  corresponds 
to  the  formula  C40H56O18,  and  melts  at  140°  C.  The  oxidizing  pig- 
ment has  a  peculiar  violet-like  odor,  at  least  in  the  case  of  plant  xan- 
thophyll,  although  Willstatter  and  Escher  did  not  notice  this  in  the 
case  of  xanthophyll  from  egg  yolk.  The  oxidized  crystals  dissolve  in 
concentrated  mineral  acids  with  a  dark  brown  color  and  in  dilute 
alkalis  with  an  intense  reddish-yellow  color. 

The  constitution  of  xanthophyll,  like  that  of  carotin,  is  unknown. 
Even  its  relation  to  carotin  is  very  puzzling.  While  the  empirical  re- 
lations between  the  two  carotinoids  suggest  that  xanthophyll  is  a  sim- 
ple oxidation  product  of  carotin,  the  behavior  of  xanthophyll  shows 
that  this  is  not  the  case.  Xanthophyll  fails  to  give  a  reaction  for 
carbonyl,  alcohol  or  acid  groups,  which  suggests  that  the  oxygen  must 
be  present  in  an  ether-like  combination.  If  this  be  accepted  as  prob- 
able it  would  indicate  that  the  carotinoids  are  not  derived  from  each 
other  but  are  rather  built  up  from  a  common  nucleus. 

Lycopin.  This  red  isomer  of  carotin  crystallizes  in  the  form  of  a 
bright  or  dark  carmine  colored,  velvety  appearing  mat  of  wax-like 
crystal  aggregates,  consisting  of  elongated  microscopic  prisms,  whose 
ends  are  usually  quite  ragged.  The  crystals  usually  obtained  from  pe- 
troleum ether  are  of  this  character  and  are  shown  in  Plate  2,  Figure 
4.  Figure  5,  Plate  2  shows  the  fine  needles  which  crystallize  from 
ether  or  from  carbon  disulfide-alcohol,  which  frequently  occur  in 
beautiful  starlike  clusters,  according  to  Monteverde  and  Lubimenko. 
The  powdered  crystals  have  a  dark  reddish-brown  color  and. melt  at 
168°-169°  C.  (corrected). 

Lycopin  crystals  are  less  soluble  than  carotin  in  all  the  carotinoid 
solvents.  Ethyl  and  especially  methyl  alcohol  are  exceptionally  poor 
solvents.  Low  boiling  petroleum  ether  dissolves  only  a  small 
amount,  10  to  12  liters  taking  up  only  1  gram.  About  3  liters  of  ether 
are  required  for  the  same  amount  of  pigment,  but  one  can  readily 
obtain  a  2  per  cent  solution  in  CS2,  and  even  stronger  solutions  in 
warm  chloroform  or  benzene.  The  crystals  are  insoluble  in  acetone 
and  glacial  acetic  acid.  They  dissolve,  however,  in  concentrated 
H2SO4  and  HNO3  with  a  deep  blue  or  purple  color,  which  is  very 
transient  in  the  case  of  HNO3. 

The  lycopin  crystals  readily  oxidize  with  bleaching,  the  maximum 
oxygen  absorption  amounting  to  about  32.5  per  cent  of  their  original 


PROPERTIES  AND  METHODS  OF  IDENTIFICATION    239 

weight.  The  oxidizing  pigment  has  a  peculiar  odor,  which  is  stated 
by  Willstatter  and  Escher  (1910)  to  be  different  from  that  of  oxidiz- 
ing carotin  or  xanthophyll,  but  js  not  described. 

When  lycopin  crystals  are  dissolved  in  a  little  CSg  and  much  ether 
and  treated  with  one-third  their  weight  of  iodine,  a  lycopin  iodide  cor- 
responding to  the  probable  formula  C40H56I2  precipitates  in  the  form 
of  dark  green,  gelatinous  flakes,  containing  34-37  per  cent  iodine. 
When  the  lycopin  crystals  are  treated  with  a  trace  of  bromine  vapor 
they  first  turn  a  vivid  green,  and  can  then  be  dissolved  in  an  excess 
of  bromine  to  form  a  colorless,  resinous  material,  insoluble  in  an- 
hydrous formic  acid,  whose  constitution  is  somewhat  difficult  to  un- 
derstand in  the  light  of  the  analyses  made  by  Willstatter  and  Escher. 
The  combined  addition  and  substitution  compound  appears  to  have 
the  constitution  CioHi^T2e,  indicating  substitution  of  12  hydrogen 
atoms  and  addition  of  14  bromine  atoms.  This  would  indicate  a  much 
greater  instability  of  the  lycopin  double  bonds  than  is  the  case  with 
carotin  which  forms  the  bromide  C4oH3eBr22,  which  shows  the  addi- 
tion of  only  two  atoms  of  bromine  and  corresponds  to  the  di-iodide 
which  the  pigment  forms. 

Fucoxanthin.  This  carotinoid  crystallizes  from  concentrated 
methyl  alcohol  in  the  forms  of  long  amber  colored  prisms,  belonging 
to  the  monoclinic  system.  The  crystals  are  shown  in  Plate  2,  Figure 
6.  The  powdered  crystals  are  brick  red.  These  crystals  contain  three 
molecules  of  methyl  alcohol.  When  freed  of  the  solvent  by  desicca- 
tion, the  crystals  are  hydroscopic.  This  water  is  difficult  to  remove, 
being  given  up  only  at  105°  C,  under  diminished  pressure.  Fucoxan- 
thin crystallizes  from  dilute  alcohol  or  acetone  in  characteristic  hexa- 
gon-shaped tablets,  containing  2  molecules  of  water  of  crystallization. 
The  addition  of  water  to  the  alcohol  or  acetone  solution  of  fucoxan- 
thin precipitates  the  pigment  as  needles  which  rapidly  change  to  the 
hexagon-shaped  hydrates.  The  anhydrous  crystals  melt  at  159.5°- 
160.5°  C.  (corrected) ,  those  containing  methyl  alcohol  about  10°  lower. 
Fucoxanthin  resembles  xanthophyll  in  its  solubilities.  One  hun- 
dred grams  of  boiling  methyl  alcohol  dissolve  1.66  grams  of  pigment, 
but  only  0.41  grams  at  0°  C.  The  crystals  are  fairly  difficultly  soluble 
in  ether,  fairly  easily  in  CS2,  and  easily  in  ethyl  alcohol.  The  pure  crys- 
tals, either  hydrates  or  methyl  alcoholates,  do  not  readily  oxidize,  but 
the  solutions  readily  bleach,  and  a  product  can  be  obtained  from  these 
colorless  solutions  which  corresponds  to  the  formula  C^oHg^Oie  or 
C40H54O14. 


240  CAROTINOIDS  AND  RELATED  PIGMENTS 

Fucoxanthin  crystals  correspond  with  the  other  carotinoids  in  dis- 
solving in  concentrated  H2SO4  and  HNO3  with  a  blue  or  purple  color, 
and  in  addition  readily  form  a  blue  oxonium  salt  with  HCl,  as  has 
already  been  described.  The  pigment  also  forms  an  iodide,  but  ap- 
parently one  containing  4  atoms  of  iodine,  instead  of  di-iodide  which 
the  other  carotinoids  form.  This  product  is  obtained  in  the  form  of 
dark  violet,  short,  pointed  prisms  with  a  copper  luster,  which  have  a 
gray  to  blue-green  color  by  transmitted  light  when  viewed  under  the 
microscope.  One  obtains  these  crystals  by  adding  the  iodine  to  an 
ether  solution  of  the  pigment.  The  crystals  are  easily  soluble  in 
chloroform  and  acetone  with  a  deep  blue  color.  They  melt  at  134°- 
135°  C.  A  bromide  of  the  pigment  does  not  appear  to  have  been 
made. 

Methods  of  Identification  in  Biological  Products 

It  is  at  times  very  desirable  to  be  able  to  identify  carotinoid  pig- 
ments in  the  plant  or  animal  tissues  in  which  they  occur.  So  far  as 
plant  tissues  are  concerned  it  is  possible  to  make  a  gross  identification 
of  carotinoids  with  certainty  and  even  to  differentiate  carotin  and 
xanthophylls  and  lycopin  with  a  reasonable  degree  of  accuracy. 
These  results  are  made  possible  because  of  the  excellent  researches  of 
Molisch  (1896),  Tammes  (1900)  and  particularly  van  Wisselingh 
(1915).  A  similar  identification  of  carotinoids  in  animal  tissues  has 
not  yet  been  devised ;  at  least  it  will  be  pointed  out  presently  how  in- 
secure the  foundation  is  upon  which  the  demonstration  of  animal 
lipochromes  (undoubtedly  carotinoids)  has  been  built.  Attention 
will  be  directed  first  to  the  possibilities  in  connection  with  plant 
tissues. 

Plant  tissues.  The  demonstration  and  identification  of  carotinoids 
in  the  plant  tissues  in  which  they  are  formed  rests  upon  a  microchemi- 
cal  crystallization  of  the  pigments  in  the  tissues  and  a  study  of  the 
effect  of  certain  solvents,  and  reagents  producing  color  reactions,  upon 
these  crystals.  As  has  already  been  pointed  out  in  a  previous  chap- 
ter, carotinoids  occur  almost  entirely  in  the  plastids  in  plant  tissues 
and  very  rarely  as  crystals  in  the  cells.  As  van  Wisselingh  has  stated, 
the  carotinoids  occur  mostly  bound  to  fluid,  fat-like,  saponifiable 
substances  or  actually  dissolved  in  them.  These  substances  are  in 
the  plastids  or  they  form  oily  drops  in  the  cells.  It  becomes  neces- 
sary, therefore,  first  to  set  the  pigments  free  from  their  union  or  solu- 
tion in  the  plastids.    It  has  been  shown  by  van  Wisselingh  that  of  the 


PROPERTIES  AND  METHODS  OF  IDENTIFICATION   241 

methods  which  have  been  proposed  for  the  microchemical  crystalliza- 
tion of  the  carotinoids  only  one  can  be  depended  upon  to  assure  this 
result,  namely  the  alkali  method  of  Molisch.  He  has  shown  that  it  is 
possible  by  this  method  to  secure  the  microcrystallization  of  all  types 
of  known  carotinoids  occurring  in  plants,  with  the  possible  excep- 
tion of  fucoxanthin.  While  van  Wisselingh  found  that  the  brown 
algse  give  excellent  crystals  it  is  not  clear  whether  these  are  fuco- 
xanthin or  the  carotin  and  xanthophyll  which  accompany  it  in  these 
plants. 

The  method  is  carried  out  as  follows.  Several  small  pieces  of  plant 
tissue  or  sections  of  the  same  (leaf,  petal,  slice  of  fruit  or  other  bulky 
tissue)  are  placed  in  100  to  200  cc.  of  alcoholic  potash  containing  40 
per  cent  alcohol  by  volume  and  20  per  cent  KOH  by  weight.  The 
tissue  and  solvent  are  placed  in  darkness  protected  from  the  air  and 
allowed  to  stand  until  the  substances  associated  with  the  carotinoids 
have  dissolved  or  become  saponified  and  the  carotinoids  present  have 
crystallized  out.  The  time  of  crystallization  will  vary  with  the  ob- 
ject from  minutes  to  months  but  can  be  greatly  speeded  up  for  the 
latter  cases  by  warming  the  preparation  for  a  few  hours  at  70°-80°  C, 
on  several  successive  days.  Van  Wisselingh  has  shown  that  crystals 
can  be  obtained  in  a  few  days  by  this  modification  which  may  require 
over  a  year  at  room  temperature.  A  piece  of  the  tissue  being  studied 
is  withdrawn  from  time  to  time  for  examination  as  to  the  progress  of 
the  reaction.  It  is  first  washed  thoroughly  with  water  and  finally 
allowed  to  rest  in  distilled  water  for  several  hours  before  preparing 
the  section  for  microscopic  examination. 

When  carotin  and  xanthophylls  are  present  the  crystals  which  will 
form  divide  themselves  into  two  general  classes  according  to  their 
color,  one  group,  probably  due  to  xanthophylls,  being  orange-yellow 
to  orange  and  the  other,  probably  due  to  carotin,  being  orange-red 
to  red.  It  is  not  safe,  however,  to  depend  upon  the  color  of  the  crys- 
tals for  determining  their  character,  because  rhodoxanthin,  if  pres- 
ent, would  undoubtedly  crystallize  in  the  red  group.  In  fact,  van 
Wisselingh  encountered  red  xanthophyll-like  crystals  in  his  investi- 
gation. 

Lycopin  does  not  form  microcrystals  in  the  Molisch  method.  A 
slight  modification,  however,  permits  their  formation,  namely,  heat- 
ing the  tissue  to  140°  C.  in  glycerol  alone  or  in  glycerol  containing 
10  per  cent  KOH.  Lycopin  forms  reddish  violet  microscopic  crystals 
under  these  conditions. 


242  CAROTINOIDS  AND  RELATED  PIGMENTS 

The  formation  of  crystals  in  plant  tissues  by  the  methods  described 
is  alone  sufficient  for  a  gross  identification  of  carotinoids.  For  veri- 
fication, however,  the  crystals  may  be  treated  either  with  strong 
H2SO4  or  bromine  water  or  with  a  solution  of  SbClg  in  25  per  cent 
HCl.  In  each  case  the  reagent  will  impart  a  blue  color  to  caroti- 
noid  crystals  of  all  types.  When  using  the  antimony  reagent  the  prep- 
aration must  first  be  placed  in  dilute  HCl.  In  the  other  cases  the  prep- 
aration may  be  placed  in  a  minimum  amount  of  water. 

The  differentiation  of  the  types  of  crystals  as  xanthophyll,  carotin 
or  lycopin,  rests  upon  two  general  tests  which  are  reasonably  accu- 
rate. (1)  Xanthophyll  crystals  dissolve  very  quickly  in  a  phenol- 
glycerol  mixture  made  up  of  3  parts  by  weight  of  phenol  and  1  pari 
by  weight  of  glycerol,  while  carotin  and  lycopin  dissolve  very  slowly 
if  at  all.  Van  Wisselingh  found  that  carotin  crystals  from  carrots 
and  lycopin  crystals  from  tomatoes  remained  untouched  by  this  re- 
agent even  after  several  days.  (2)  Xanthophyll  crystals  give  a  quick 
blue  color  when  treated  with  75  per  cent  H2SO4  while  carotin  and 
lycopin  crystals  require  a  stronger  H2SO4  to  color  their  crystals  blue, 
or  at  least  to  do  so  quickly. 

Animal  tissues.  The  possibility  of  a  microchemical  demonstration 
of  carotinoids  in  animal  tissues  rests  at  the  present  time  on  the  as- 
sumption that  the  methods  which  have  been  used  for  identifying  lipo- 
chromes  in  such  tissues  are  in  reality  methods  for  detecting  caroti- 
noids, and  are,  moreover,  specific  for  these  pigments.  Let  us  see 
whether  these  assumptions  are  justified. 

Two  methods  have  been  used  rather  generally  for  detecting  lipo- 
chromes  in  sections  of  animal  tissues.  One  has  been  the  application 
of  the  so-called  specific  color  reactions  with  concentrated  H2SO4  and 
HNO3  and  with  iodine-potassium-iodide  solution;  the  other  has  been 
the  reaction  of  the  pigments  towards  certain  fat  stains,  particularly 
Scarlet  Red,  Sudan  III  and  osmic  acid. 

Carotinoid  pigments  are  encountered  in  animal  tissues  both  as  intra- 
cellular and  intercellular  substance,  generally  in  more  or  less  gran- 
ular or  amorphous  condition  but  also  coloring  what  appears  to  be  true 
fat  globules.  The  possibility  is  also  not  excluded  that  they  may  oc- 
cur in  the  tissues  bound  to  protein  as  they  at  times  occur  in  the  blood. 
Since  carotinoids  dissolve  readily  in  liquid  fats,  one  may  also  expect 
to  find  fats  at  times  dissolved  in  carotinoids.  It  is  therefore  an  open 
question  whether  true  lipochromes  (carotinoids)  ever  occur  in  ani- 
mal tissues  in  a  pure  condition.     Since  lipochrome  is  never  encoun- 


PROPERTIES  AND  METHODS  OF  IDENTIFICATION   243 

tered  in  a  crystalline  condition  in  such  tissues,  as  it  has  been  stated 
to  occur  in  plants,  the  balance  of  the  argument  is  at  present  against 
the  occurrence  of  the  pure  pigments. 

With  regard  to  the  lipochrome  color  reactions  the  results  which 
have  been  secured  are  not  encouraging  as  to  their  applicability  to  the 
carotinoids  which  occur  in  animal  tissues.  The  chemistry  of  the 
blue  color  reaction  with  con.  HgSO^,  which  is  given  by  a  number  of 
aromatic  substances  besides  the  carotinoids,  is  not  known.  A  positive 
reaction  with  this  reagent  can  not  therefore  be  regarded  as  conclusive 
proof  of  the  presence  of  carotinoids,  although  it  would  indicate  this 
possibility.  This  reaction,  however,  fails  completely  in  the  presence 
of  glycerides,  and  may  be  vitiated  even  by  the  presence  of  other 
lipoids  which  are  attacked  by  strong  sulfuric  acid.  It  can  not  be  used 
at  all  for  detecting  carotinoids  dissolved  in  fats.  A  negative  test 
on  even  more  suitable  material  is  not  necessarily  conclusive;  for 
example,  Sehrt  (1904)  discredited  the  corpus  luteum  pigment  as  a 
lipochrome  because  it  was  colored  only  a  faint  blue  by  H2SO4,  and 
sometimes  not  at  all.  The  reaction  with  H2SO4,  therefore,  has  only 
a  very  limited  application  to  carotinoids  which  may  be  encountered 
in  histological  sections  of  animal  tissues,  at  least  under  the  conditions 
in  which  it  has  been  employed  up  to  the  present  time. 

Practically  the  same  conclusion  must  be  drawn  for  the  use  of  the 
iodine  reaction.  The  cause  of  this  reaction  is  the  blue  iodine  deriva- 
tive which  is  formed  by  all  the  known  carotinoids.  The  reaction 
was  discovered  by  Schwalbe  (1874)  as  an  apparently  typical  pig- 
ment reaction  for  the  colored  oil  drops  in  the  retina  of  certain  ani- 
mals. Since  that  time  it  has  been  generally  held  that  animal  pig- 
ments which  fail  to  give  the  iodine  reaction  are  not  typical  lipo- 
chromes.  However,  even  this  reaction,  which  possesses  a  firm  chemi- 
cal basis,  has  frequently  failed  for  pigments  which  we  now  know 
to  be  true  carotinoids.  For  example,  Kiihne  (1878)  failed  to  secure 
the  reaction  with  egg  yolk  pigment,  even  after  isolation,  and  Sehrt 
(1904)  found  that  the  corpus  luteum  pigment  only  occasionally  re- 
acted. 

These  results  seem  very  discouraging.  We  have  already  seen,  how- 
ever, that  no  great  difficulty  attends  the  use  of  color  reactions  in  the 
microchemical  identification  of  carotinoids  in  plant  tissues.  The 
author  is  not  aware  of  any  attempt  to  apply  the  Molisch  microchemi- 
cal method  of  crystallization  of  carotinoids  to  animal  tissues.  Un- 
fortunately the  high  concentration  of  alkali  in  the  Molisch  reagent 


244  CAROTINOIDS  AND  RELATED  PIGMENTS 

would  undoubtedly  disintegrate  animal  tissues  before  the  pigment 
could  crystallize  out.  It  would  be  well  worth  the  effort,  however, 
if  a  microchemical  crystallization  method  could  be  devised  which 
would  be  applicable  to  animal  tissues. 

The  conception  of  a  constant  association  of  pigment  with  fat  which 
is  suggested  by  the  term  lipochrome  was  no  doubt  in  a  measure  re- 
sponsible for  the  introduction  of  fat  stains  for  demonstrating  the 
presence  of  such  pigments  in  animal  tissues.  The  writer  has  not 
made  a  thorough  study  of  the  history  of  the  use  of  this  technic,  since 
the  matter  is  not  of  great  importance.  It  is,  therefore,  a  little  dif- 
ficult to  state  whether  the  use  of  fat  stains  began  with  the  idea  of  dem- 
onstrating that  a  pigment  in  question  was  actually  associated  with  fat 
and  therefore  a  true  "lipochrome,"  or  whether  their  use  was  suggested 
solely  by  the  term  itself  or  by  the  statements  encountered  here  and 
there  in  the  literature  on  plant  lipochromes  that  one  of  the  fat  stains 
(usually  osmic  acid)  imparted  its  characteristic  color  to  the  pigment. 
Whatever  the  origin  of  their  use  may  have  been  it  is  obvious  that  the 
present  conception  of  the  action  of  the  fat  stains,  as  shown  by  con- 
sulting the  modern  handbooks  on  biochemistry  and  pathology,  is  that 
they  stain  the  pigments  themselves.  For  example.  Wells  (1918) 
states  that  the  lipochromes  "are  characterized  by  staining  by  such 
fat  stains  as  Sudan  III  and  Scarlet  Red,  and  usually,  but  not  con- 
stantly, by  osmic  acid";  and  Herxheimer  (1913)  makes  practically 
the  same  statement,  without,  however,  making  any  reservations  with 
respect  to  osmic  acid. 

As  far  as  the  true  carotinoids  are  concerned  this  conception  rests 
upon  the  uncertain  assumption  that  these  pigments  are  actually 
stained  by  such  dyes  as  Sudan  III,  Scarlet  Red  and  osmic  acid.  It 
is  possible  that  such  is  the  case,  but  unfortunately  the  matter  has 
never  been  subjected  to  an  experimental  study;  and  until  we  have 
further  proof  of  the  action  of  these  and  other  fat  dyes  upon  the 
pure  pigments  it  is  not  possible  to  state  definitely  that  a  positive  stain 
with  a  fat  dye  is  a  positive  test  for  pigment  of  the  carotinoid  (lipo- 
chrome) type.  In  fact,  there  is  evidence  which  indicates  that  a.  posi- 
tive stain  with  a  fat  dye  is  merely  a  test  for  the  lipoid  with  which 
the  pigment  is  associated. 

Neumann  (1902)  states  that  when  the  fat  cells  of  the  bone  marrow 
and  sex  glands  of  frogs  have  become  completely  atrophied  through 
inanition  (or  during  hibernation)  the  lipochrome  which  remains  no 
longer  takes  the  osmic  acid  stain,  but  still  gives  the  reaction  with 


PROPERTIES  AND  METHODS  OF  IDENTIFICATION  245 

iodine.  Dolley  and  Guthrie  (1919)  have  studied  carotinoids  in  animal 
tissues  by  means  of  fat  stains  with  results  which  bear  on  this  ques- 
tion. They  observed  that  amorphous  deposits  of  what  appeared  to 
be  pure  lipochrome  in  both  animal  and  plant  (carrot)  tissues  still 
stained  with  Sudan  III  and  Scarlet  Red  after  the  pigment  granules 
had  become  bleached  through  oxidation.  This  fact  does  not  prove 
that  the  pigment  granules  were  not  pure  pigment,  but  it  does  throw 
doubt  upon  this  conclusion.  Kreibich  (1920)  takes  the  view  that  the 
lipochromes  in  animal  tissues,  which  he  calls  sudanophiles,  are  united 
with  alcohol  insoluble  lipoids. 

Dolley  and  Guthrie  (see  also  Palmer  and  Kempster  (1919b))  made 
the  interesting  observation  that  Nile  blue  (used  either  as  the  hydro- 
chlorate  or  sulfate  in  1-10,000  aqueous  solution  or  as  a  stronger  so- 
lution in  65  per  cent  acetone)  when  used  as  a  progressive  stain, 
''particularizes  the  lipochrome  first  as  a  deep  blue,"  but  stains  neu- 
tral fat  a  salmon  pink  even  in  the  presence  of  lipochrome.  The  blue 
stain  was  found  to  occur  for  the  amorphous  carotin  granules  in  the 
frozen  carrot  section,  and  for  the  amorphous  xanthophyll  granules 
and  minute  pigment  globules  in  the  stratum  corneum  of  the  chicken 
skin,  while  the  salmon  pink  color  occurred  for  lard  stained  deeply 
with  carotin  from  carrots,  for  chicken  fat  highly  colored  with  xan- 
thophyll, for  the  globules  in  colostrum  milk  fat,  deeply  colored  with 
carotin,  and  for  the  fat  globules  in  the  natural  emulsion  of  the  deep 
yellow  (xanthophyll  colored)  yolk  of  the  hen's  egg.  Dolley  and 
Guthrie  later  (1921)  found  that  the  lipochrome  granules  of  the  heart 
muscle  stained  blue  with  the  dye,  although  in  their  earlier  (1919) 
work  they  were  unable  to  secure  positive  differentiation  of  lipo- 
chrome and  fat  in  nerve  cells  by  this  method. 

These  findings  appear,  at  first  sight,  to  be  a  definite  advance  in  the 
technic  of  demonstrating  carotinoids  in  animal  tissues.  This  conclu- 
sion is  weakened,  however,  by  the  fact  that  Smith  (1907)  showed  that 
Nile  blue  differentiates  fatty  acids  from  neutral  fats  in  the  same  man- 
ner that  it  appears  to  differentiate  carotinoids  from  neutral  fats,  fatty 
acids  staining  blue  and  neutral  fat  red.^  This  fact  alone,  however, 
would  not  necessarily  disprove  the  supposition  that  carotinoids  may 
act  like  fatty  acids  towards  Nile  blue,  although  it  must  be  admitted 

»Herxheimer  (Abderhalden's  Handbuch  der  Biologisclien  Arbeltsmetnoden,  viilj  part 
1,  208)  states  that  this  fact  has  been  confirmed  by  Escher  (Korrbl.  f.  Schweizer  Arzte, 
Jf9,  1919)  and  by  Boeminghaus  (2iegler's  Beitrage,  67,  532,  1920)  who  found  also  that 
cholesterol  esters  of  fatty  acids  stain  red  like  neutral  fat,  and  many  other  lipoids  take 
a  mixed  blue  and  red  color. 


246  CAROTINOIDS  AND  RELATED  PIGMENTS 

that  the  chemistry  of  the  blue  stain  with  Nile  blue  argues  against 
this  supposition.  As  stated  by  Smith  the  simultaneous  staining  of 
fatty  acids  and  neutral  fat  by  Nile  blue  is  due  to  the  fact  that  this 
dye  is  a  mixure  of  a  strongly  basic  oxazine  which  reacts  readily  with 
fatty  acids  to  form  blue  soaps,  and  a  weakly  basic  oxazone  which 
dissolves  readily  in  neutral  fats  and  fat  solvents.  When  it  is  remem- 
bered that  none  of  the  carotinoids  have  acid  properties  it  may  be 
argued  that  the  blue  stain  imparted  to  carotinoid  pigment  granules  in 
the  writer's  and  in  Dolley  and  Guthrie's  experiments  merely  indi- 
cates the  acid  character  of  the  lipoid  with  which  the  pigment  was  as- 
sociated. This  argument  would  have  to  be  accepted  as  conclusive  if 
the  oxidized  pigment  granules  should  be  found  to  retain  their  prop- 
erty of  taking  the  blue  stain  with  the  Nile  blue  oxazine  like  they  have 
been  observed  to  do  with  Sudan  III  and  Scarlet  Red.  On  the  other 
hand,  if  it  should  be  found  that  the  oxidized  pigment  granules  in 
plant  or  animal  tissues  no  longer  take  the  blue  stain  with  Nile  blue 
there  would  be  strong  basis  for  believing  that  the  oxazine  base  in  the 
dye  is  specific  for  carotinoids  as  well  as  for  fatty  acids.  Certainly 
it  would  not  be  unreasonable  to  assume  that  the  profound  changes 
which  undoubtedly  occur  in  the  chemical  characters  of  the  carotinoids 
during  their  oxidation  also  alter  their  relation  towards  dyes. 

Hueck  (1912)  has  stated  that  lipochrome  in  animal  tissue  still 
stains  blue  with  Nile  blue  after  oxidation  with  hydrogen  peroxide. 
At  the  writer's  suggestion  Dr.  Dolley  has  investigated  this  point  more 
exhaustively  using  frozen  sections  of  carrot  tissue,  with  the  result^ 
that  Hueck's  observation  is  confirmed.  Complete  oxidation  with 
hydrogen  peroxide  and  sunlight  or  careful  oxidation  with  ferric  chloride 
fails  to  destroy  the  ability  of  the  visible,  bleached  pigment  granules  to 
take  the  blue  oxazine  base  from  the  Nile  blue  dye.  It  thus  appears 
impossible  to  differentiate  between  carotinoid  pigment  and  fatty  acids, 
although  these  can  be  distinguished  from  neutral  fat  or  esters  by  the 
Nile  blue  dye. 

The  effect  of  ferric  chloride  on  the  carotinoids  is  alone  of  some 
value  in  indicating  the  presence  of  carotinoids  in  animal  tissues. 
While  it  is  not  possible  to  obtain  the  green  color  reaction  when  work- 
ing with  tissue  sections,  on  account  of  the  fact  that  the  color  reaction 
is  in  the  reagent  itself,  pigment  granules  which  are  readily  oxidized 
(bleached)  by  this  reagent  may  be  suspected  to  be  carotinoid  in  na- 
ture.    Dolley   and  Guthrie   found  that  a  strong  solution  of  ferric 

*  Personal  communication. 


PROPERTIES  AND  METHODS  OF  IDENTIFICATION   247 

chloride  in  50  per  cent  alcohol  was  especially  suitable  for  this  pur- 
pose. Treatment  of  sections  showing  carotinoid  granules  with  hydro- 
gen peroxide  for  24  to  48  hours  apparently  effected  the  same  result, 
but  Dr.  Dolley's  recent  communication  indicates  that  the  oxidation 
is  not  so  thorough  as  with  the  ferric  chloride  unless  the  peroxide  is 
supplemented  with  strong  sunlight.  There  is  also  the  tacit  assumption 
among  pathologists  that  sections  which  give  up  their  pigment  to  fat 
solvents  contain  lipochromes.  This,  of  course,  can  only  be  considered 
contributory  evidence. 

Summary 

It  is  impossible  to  summarize  adequately  in  a  few  words  the  facts 
presented  in  this  chapter  describing  in  detail  the  chemical  and  physi- 
cal properties  of  the  several  carotinoids  both  in  crystalline  form  and 
also  when  in  solution  in  various  solvents. 

It  may  be  pointed  out,  however,  that  the  properties  of  impure 
solutions  of  the  individual  carotinoids  when  freshly  prepared  are  suf- 
ficiently characteristic  for  their  identification  without  resorting  to  the 
tedious  process  of  isolating  the  pigments  in  pure  crystalline  form.  The 
characteristic  properties  which  may  be  employed  for  this  purpose  in- 
clude color,  spectroscopic  absorption  bands,  relative  solubility  in  al- 
cohol and  petroleum  ether  and  adsorption  affinity  towards  finely  di- 
vided agents  like  CaCOg. 

It  is  fortunately  possible  to  identify  carotinoids  in  general  in  plant 
issues  through  a  microchemical  crystallization  method.  It  is  possi- 
ble, also,  to  roughly  differentiate  these  crystals  into  groups,  such  as 
carotin,  xanthophyll  and  lycopin-like  pigments,  by  means  of  the  ef- 
fect of  a  phenol-glycerine  solvent  on  the  crystals  and  the  rapidity 
with  which  they  respond  to  a  color  reaction  with  sulfuric  acid  of 
different  strengths. 

The  microchemical  demonstration  of  carotinoids  in  animal  tissues 
does  not  rest  on  a  very  adequate  basis.  Recent  work,  however,  indi- 
cates that  although  it  is  not  possible  to  differentiate  between  carotinoid 
pigment  and  fatty  acids,  these  can  be  distinguished  from  neutral  fat 
and  esters  by  means  of  their  characteristic  staining  reaction  with  Nile 
blue,  the  former  staining  blue  and  the  latter  some  shade  of  pink. 


Chapter  X 
Quantitative  Estimation  of  Carotinoids 

The  small  amount  of  carotinoids  in  plant  and  animal  tissues,  to- 
gether with  the  difl&culty  of  securing  the  pigments  free  from  color- 
less impurities  as  well  as  the  great  ease  with  which  the  pigments  oxi- 
dize, forbid  their  quantitative  estimation  by  a  gravimetric  method. 
The  great  intensity  of  the  carotinoid  pigments  and  their  ready  solu- 
bility in  certain  organic  solvents  naturally  suggests  the  possibility  of 
their  quantitative  estimation  by  colorimetric  methods.  The  methods 
which  have  been  proposed  have,  in  fact,  been  devised  on  this  basis. 

Estimation  of  Carotin  and  Xanthophyll 

Arnaud  (1887)  was  the  first  to  propose  a  colorimetric  method  for 
the  quantitative  estimation  of  carotin  in  plant  tissues.  The  method 
was  based  on  his  observation  that  air  dried,  or  especially  vacuum 
dried  leaves  (leaves  dried  in  an  oven  even  at  low  temperature  cannot 
be  used,  according  to  Arnaud)  do  not  give  up  any  of  their  chlorophyll 
when  allowed  to  remain  in  contact  with  low  boiling  petroleum  ether, 
but  permit  all  the  carotin  to  be  extracted,  A  further  essential  fea- 
ture of  his  method  was  based  on  the  observation  (a  single  experi- 
ment only  is  reported)  that  the  color  of  carbon  disulfide  solutions  of 
carotin  is  directly  proportional  to  the  amount  of  carotin  present. 
With  these  observations  as  a  basis  Arnaud  proceeded  as  follows. 

Twenty  gram  quantities  of  air-dried  or  vacuum-dried,  powdered 
leaves  were  shaken  up  with  1  liter  of  cold  petroleum  ether  in  a  stop- 
pered flask  for  a  period  of  10  days.  The  extract  was  filtered  off  and 
exactly  100  cc.  evaporated  to  dryness.  The  residue  was  taken  up  in 
exactly  100  cc.  of  carbon  disulfide  and  compared  in  a  Dubosque  col- 
orimeter with  a  standard  0.001  per  cent  solution  of  carotin  in  carbon 
disulfide.  It  is  stated  that  the  colorimeter  was  modified  slightly  to 
prevent  the  volatilization  of  the  solvent  and  that  blue  glasses  were 
inserted  to  improve  the  sensitiveness  of  the  instrument,  but  the  de- 
tails in  regard  to  these  modifications  are  not  given.    The  data  which 

248 


QUANTITATIVE  ESTIMATION  OF  CAROTINOIDS     249 

Arnaud  reports  indicate  that  he  set  his  standard  carotin  solution  at 
19  mm.  depth  and  compared  his  unknown  solutions  with  this  arbi- 
trary standard. 

Arnaud  (1889)  used  this  method  for  the  quantitative  analysis  of 
the  carotin  content  of  the  air-dried  leaves  of  a  large  number  of  plants. 
These  data  are  shown  in  Table  17.  Arnaud  expresses  his  confidence 
in  their  accuracy  within  1  to  2  mg.  of  carotin  for  100  grams  of  dried 
leaves.  The  principal  criticisms  which  can  be  made  of  this  method 
are,  (1)  the  necessity  of  having  a  supply  of  pure  carotin  on  hand  for 
making  up  the  standard  solution,  (2)  the  failure  to  show  that  the 
method  insures  the  complete  extraction  of  the  carotin,  (3)  the  pos- 
sibility that  some  xanthophyll  may  be  extracted  along  with  the  caro- 
tin, and  (4)  the  necessity  of  evaporating  the  petroleum  ether  extracts 
to  dryness  before  dissolving  in  the  standard  solvent,  this  operation 
greatly  enhancing  the  opportunities  for  oxidation  and  loss  of  pig- 
ment. 

Table  17.    Carotin  Content  of  Air-Dried  Leaves  (Arnaud's  Method) 

Name  of  plant  Date  of  Carotin 

sample  content 

mg.inlOO  gms. 

Rape  {Brassica  oleijera) June  1  1897 

Violet  {Viola  odorata) May  23  124.0 

Linden  {Tilia  platyphylla) May  11  79.1 

Maple  (Acer  pseudo-platanus) June  15  190.0 

Sycamore  (Acer  platanoides) June  15  178.0 

Grape  (Vitis  vinijera) July  12  200.0 

Wild  grape  (Cissus  quinquejolia) May  25  145.4 

Chestnut   (Aesculus  hypocastanum) May  6  118.8 

Bean  (Phaseolus  vulgaris) June  18  178.8 

Pea  {Pisum  sativum) May  27  177.0 

Acacia  {Robinia  pseudo-acacia) June  8  209.0 

Peach  {Persica  vulgaris) June  15  114.0 

Red  currant  {Ribes  rubrum) May  21  105.5 

Ivy   (Hedera  Helix) May  15  50.9 

Periwinkle   {Vinca  Major) May  25  130.0 

Olive   (Olea  Europaea) July  16  75.0 

Potato  iSolanum  tuberosum) July  21     ~  190.0 

Tobacco  (Nicotinia  tabacum) Aug.  4  178.8 

Stramonium  (Datura  stramonium) July  20  177.0 

Spinach    (Spinacia  inermis) June  1  160.0 

Beet  (Beta  vulgaris) July  12  183.0 

Hemp  (Cannabis  sativa) June  18  215.9 

Box  tree  (Buxu^  sempervirens)   June  4  86.9 

Stinging  nettle  (Urtica  dioica) May  2  _         171.7 

Walnut  (Juglans  regia) May  19         '         118.8 

Yew  tree  ( Taxus  baccata) June  4  167.6 

Wheat  (Triticum  vulgare) June  4  167.6 

Grass  (Lolium  perenne) April  18  106.3 

Fern  (Pteris  aquilina) June  1  116.8 


250  CAROTINOIDS  AND  RELATED  PIGMENTS 

Arnaud  made  an  interesting  study  of  the  seasonal,  variations  in  the 
carotin  content  of  green  leaves,  using  the  stinging  nettle  and  chest- 
nut leaves  as  the  source  of  his  material.  He  found  that  the  maximum 
carotin  content  (on  the  dry  basis)  occurred  at  the  time  of  the  flower- 
ing of  the  plants,  and  that  it  diminished  regularly  with  the  growth 
of  the  leaves.  Thus,  in  the  case  of  the  nettle,  a  carotin  content  of 
172  mgs.  per  100  grams  of  dried  leaves  was  noted  on  May  2,  but  this 
had  decreased  to  about  100  mg.  by  the  middle  of  July.  In  a  com- 
parison between  etiolated  and  green  leaves  of  the  same  plant,  Arnaud 
found  that  the  carotin  content,  on  the  dry  matter  basis,  increased 
about  5  times  during  the  formation  of  the  chlorophyll. 

Kohl  (19021)  used  Arnaud's  method  for  determining  the  carotin 
content  of  the  leaves  of  a  few  plants,  but  secured  somewhat  lower  re- 
sults. His  values  for  spinach  and  stinging  nettle  leaves  were  about 
half  those  reported  by  Arnaud,  and  for  grass  about  70  per  cent  of 
Arnaud's  value.  These  low  results  may  have  been  due,  however,  to 
the  fact  that  Kohl  apparently  ignored  Arnaud's  precaution  and  dried 
his  material  at  100°  C. 

Monteverde  and  Lubimenko  (1913a)  have  devised  a  spectro-colori- 
metric  method  for  the  quantitative  estimation  of  carotin,  as  well  as 
xanthophylls  and  chlorophyll,  in  green  leaves.  The  writer  has  not 
been  able  to  secure  a  clear  translation  of  this  method  which  has  been 
published  only  in  Russian.  In  general,  however,  the  method  appears 
to  be  based  on  the  extraction  of  all  the  pigments  from  fresh  leaves, 
0.1  gram  quantities,  by  grinding  in  a  mortar  with  alcohol.  Measured 
quantities  of  the  extract  are  then  treated  with  strong  Ba(0H)2  solu- 
tion to  throw  down  all  the  pigments.  After  standing  for  some  hours, 
the  precipitate  is  filtered  off  and  extracted  completely  with  abso- 
lute alcohol,  which  is  said  to  take  out  only  the  carotinoids.  These 
are  fractionated  by  the  Kraus  method  between  80  per  cent  alcohol 
and  petroleum  ether,  and  these  fractions  compared  in  the  spectro- 
colorimeter  with  standard  0.001  per  cent  carotin  and  xanthophyll  so- 
lutions. By  keeping  all  extracts  in  definite  volumes  the  data  can  be 
calculated  back  to  the  quantity  of  pigments  in  the  plant  tissues  exam- 
ined. The  feature  of  the  spectro-colorimetric  method  is  the  compari- 
son of  the  solutions  on  the  basis  of  the  depth  of  unknown  solution 
required  to  give  an  absorption  spectra  of  equal  intensity  as  the  stand- 
ard. The  authors  found  that  the  first  faint  appearance  of  absorp- 
tion bands  for  the  standard  solutions  gave  a  more  sensitive  cotnpari- 


QUANTITATIVE  ESTIMATION  OF  CAROTINOIDS     251 

son  than  stronger  bands,  and  that  this  was  secured  at  a  depth  of  3  cm. 
for  the  carotin  and  xanthophyll  standards. 

Using  the  above  method  Monteverde  and  Lubimenko  determined 
the  carotin  (and  xanthophyll)  content  of  a  number  of  plants.  The 
data  are  given  in  Table  18.  The  results  are  striking  in  so  far  as  the 
low  content  of  carotin  is  concerned  in  comparison  with  the  results  se- 
cured by  Arnaud.  The  writer  is  not  convinced  that  absolute  alcohol 
will  give  a  quantitative  extraction  of  carotin  from  the  baryta-chloro- 
phyll complex  obtained  by  this  method.  The  data  in  Table  18  are 
of  interest  also  in  showing  quite  wide  variations  between  the  relative 
amounts  of  carotin  and  xanthophyll  in  the  different  plants. 

Table  18.    Carotinoid  Content  of  Dry  Green  Leaves  (Lueimenko's  Method) 

Carotin  Xanthophyll 

Name  of  plant                                                             content  content 

mg.  per  mg.  per 

100  gms.  100  gms. 

Thuja  orientalis  (arbor  vitse) 20.8  131.7 

Viburnum  Tinus 47.9  154.3 

Luffa  gigantia   61.5  354.6 

Albizzia  Julibrissin    66.7  280.9 

Ruta  graveolens   94.4  387.6 

Ailanthus   glandulosa    72.7  263.3 

Clematis  vitalba 100.6  406.5 

Hyssopus  officinalis   108.1  355.6 

Rubus^  caesius   106.0  396.8 

Arundinaria  japonica    106.1  350.0 

Willstatter  and  Stoll  (1913)  have  described  in  great  detail  a  colori- 
metric  method  for  the  quantitative  estimation  of  carotin  and  xantho- 
phylls  which  appears  to  give  very  accurate  results.  The  method  as 
given  is  intended  to  be  used  for  green  plant  tissues.  For  convenience 
in  understanding  the  details  the  method  may  be  divided  into  several 
parts,  as  follows:  (1)  Preparation  of  the  material,  (2)  extraction  of 
the  pigments,  (3)  removal  of  the  chlorophyll,  (4)  separation  of  the 
carotinoids,  (5)  colorimetric  comparison  of  the  carotinoids  with  stand- 
ard solutions. 

Preparation  of  the  material.  Forty  grams  of  fresh  leaves  are 
placed  in  a  mortar  (diam.  25  cm.)  with  50  cc.  of  40  per  cent  acetone 
and  macerated  quickly  with  0.5  gram  of  quartz  sand.  One  hundred 
cc.  of  30  per  cent  acetone  are  then  poured  over  the  apparently  dry 
mass  and  the  whole  mixed  for  a  few  minutes.  The  extract  and  leafy 
material  are  then  transferred  to  a  suction  filter  containing  a  thin  layer 
of  talc,  and  the  extract  sucked  away.  After  sucking  dry,  the  material 
on  the  filter  is  washed  with  100-200  cc.  of  30  per  cent  acetone  in  small 


252  CAROTINOIDS  AND  RELATED  PIGMENTS 

portions,  or  until  the  filtrate  is  colorless.  According  to  Willstatter 
and  Stoll  the  grinding  and  preliminary  extraction  require  15  to  30 
minutes.    The  preliminary  extracts  are  discarded. 

Extraction  of  the  pigments.  The  dry  mixture  of  leafy  material 
and  sand  on  the  filter  is  carefully  loosened  with  a  spatula  and  macer- 
ated for  a  few  minutes  with  a  small  amount  of  pure  acetone,  and  the 
acetone  quickly  sucked  away.  This  is  repeated  until  the  acetone 
comes  through  colorless,  at  which  time  the  powder  on  the  filter  will 
also  be  colorless.  The  total  volume  of  acetone  extract  will  vary  be- 
tween 400  and  600  cc,  depending  on  the  kind  of  leaves  used. 

Removal  of  the  chlorophyll.  The  green  acetone  extract  is  divided 
into  parts  of  100-200  cc,  to  each  of  which  200-250  cc.  of  ether  are 
added  and  the  acetone  washed  out  with  distilled  water.  The  ether 
fractions  are  now  combined,  dried  over  anhydrous  sodium  sulfate  and 
filtered  through  a  dry  filter  into  a  200  cc.  graduated  flask  which  is 
filled  to  the  mark  with  dry  ether. 

One  hundred  cc.  of  this  ether  are  saponified  with  2  cc.  of  concentrated 
methyl  alcohol  solution  of  KOH  by  shaking  carefully  by  hand  and 
then  in  a  shaking  machine  for  30  minutes.  After  standing  a  little 
while  the  ether  is  usually  a  pure  yellow  color,  but  if  it  still  shows 
a  red  fluorescence  the  shaking  is  continued,  if  necessary  with  the  ad- 
dition of  more  alkali.  After  complete  saponification  of  the  chloro- 
phyll the  ether  solution  is  decanted  from  the  alkali-chlorophyllines 
into  a  small  separatory  funnel  and  the  chlorophyll  salts  washed  gently 
with  ether.  In  order,  however,  to  completely  free  the  precipitate  of 
occluded  xanthophylls  30  cc.  more  ether  are  added  to  the  alkaline 
material,  the  mixture  shaken,  and,  after  adding  water,  allowed  to 
stand  until  the  emulsion  has  broken.  If  necessary  this  is  repeated 
with  fresh  ether. 

The  ethereal  solutions  thus  obtained  are  washed  with  water  to  which 
a  little  methyl  alcohol  solution  of  KOH  is  added  in  order  to  sepa- 
rate traces  of  chlorophylline  and  small  amounts  of  brown  acid  organic 
substances.  The  ether  is  finally  washed  twice  with  pure  water  and 
evaporated  to  a  volume  of  a  few  cubic  centimeters  in  a  vacuum  dis- 
tillation flask  at  room  temperature. 

Separation  of  the  carotinoids.  The  concentrated  ether  solution  of 
carotinoids  in  the  vacuum  distillation  flask  is  washed  into  a  separatory 
funnel  with  80  cc.  of  petroleum  ether,  the  flask  being  washed  out 
finally  with  a  little  ether.  This  solution  is  now  mixed  successively 
with  100  cc.  of  85  per  cent  methyl  alcohol,  100  cc.  of  90  per  cent 


QUANTITATIVE  ESTIMATION  OF  CAROTINOIDS     253 

methyl  alcohol  and  twice  with  50  cc.  of  92  per  cent  methyl  alcohol. 
The  last  extract  is  generally  colorless;  if  not,  another  extraction  is 
made  with  92  per  cent  alcohol. 

The  methyl  alcohol  extracts  contain  the  xanthophylls.  They  are 
also  free  from  carotin,  according  to  Willstatter  and  Stoll,  The  com- 
bined methyl  alcohol  extracts  are  now  mixed  with  130  cc.  of  ether  and 
the  pigments  transferred  to  the  ether  by  a  slow  addition  of  water. 
The  ether  solution  of  the  xanthophylls  thus  obtained  and  the  petro- 
leum ether  solution  of  carotin  are  freed  from  methyl  alcohol  by  wash- 
ing twice  with  water.  The  solutions  are  then  filtered  through  dry 
filters  into  100  cc.  graduated  flasks,  the  solutions  cleared  up  by  the 
addition  of  a  few  drops  of  absolute  alcohol  and  the  flasks  filled  to 
the  mark  with  ether  and  petroleum  ether  respectively. 

Colorimetric  comparison  with  standard  solutions.  The  carotin  and 
xanthophyll  fractions,  representing  20  grams  of  fresh  leaves,  are  now 
ready  for  comparison  with  standard  solutions  in  a  colorimeter.  For 
this  purpose  one  can  use  either  pure  carotin  or  xanthophyll  solutions 
in  petroleum  ether  and  ether,  respectively,  or  their  color  equivalents, 
namely,  0.25  per  cent  alazirin  in  chloroform,  or  a  0.2  per  cent  aqueous 
solution  of  KaCraOy.  The  pure  pigment  standards  are  not  satisfactory 
because  of  their  instability,  the  xanthophyll  standard,  especially,  fad- 
ing quite  rapidly.  The  dichromate  solution  is  especially  well  suited 
for  a  substitute  because  a  standard  solution  once  made  will  keep  in- 
definitely. It  is  necessary,  however,  to  know  its  color  value  in  terms 
of  the  pure  carotinoid  pigment  solutions.  Using  5  x  10~^  molar  so- 
lutions of  carotin  and  xanthophyll,  respectively,  equivalent  to  0.0268 
per  cent  carotin  solution  and  0.0284  per  cent  xanthophyll  solution,  Will- 
statter and  Stoll  found  the  following  relations  to  exist  between  the 
standard  0.2  per  cent  KgCrgOy  solution  and  the  carotinoids. 

100  mm.  carotin  solution  equals  101  mm.  KzCraOi  solution 


50 
25 
100 
50 
25 


41 

"  "  "        19 

xanthophyll  "  "        72 

li  «  t(        27 

«  "  «        j4 


In  Willstatter  and  Stoll  experiments  the  standard  carotinoid  solu- 
tions only  were  apparently  used.  The  standard  solutions  were  always 
set  at  a  depth  of  100  mm.  and  the  height  of  the  unknown  adjusted 
until  the  colors  matched.  Readings  were  then  taken  with  the  cups 
reversed  in  the  colorimeter  and  the  results  averaged.  A  Wolff  colori- 
meter was  used  by  these  investigators  but  a  Dubosque  or  Kober 


254  CAROTINOIDS  AND  RELATED  PIGMENTS 

colorimeter  should  serve  the  purpose  just  as  well.  The  writer  has 
found  the  Kober  colorimeter  very  satisfactory,  using  daylight  as  the 
source  of  illumination  and  the  black  glass  cups  with  the  colorless,  op- 
tical glass  bottoms  for  holding  the  solutions. 

Calling  he  the  height  of  the  unknown  solution  required  to  match  the 
color  of  100  mm.  of  standard  carotin  solution  and  h^  the  height  of  the 
unknown  solution  required  to  match  the  color  of  100  mm.  of  stand- 
ard xanthophyll  solution  the  amount  of  carotin  or  xanthophyll  in  1 
kg.  of  fresh  leaves  can  be  calculated  from  the  amount  obtained  from 
20  grams  by  the  method  of  Willstatter  and  Stoll  by  means  of  the 
following  formulae: 

Carotin  equals   50  x  0.00536  x -jr-x  -r— '  and 
Xanthophyll    equals   50  x  0.00568  X;r-  x  -r— - 

If  the  standard  potassium  dichromate  solutions  have  been  used  in 
place  of  the  pure  carotin  and  xanthophyll,  the  same  formulae  are 
used  because  the  dichromate  is  used  at  a  depth  of  color  corresponding 
to  100  mm.  of  the  carotinoid  solutions.  These  need  not  necessarily  be 
set  at  the  values  corresponding  to  100  mm.  of  the  carotinoid  solutions, 
but,  if  desired,  can  be  set  at  the  values  corresponding  to  50  or  25  mm. 
of  the  standard  carotinoid  solutions.  In  fact,  the  writer  believes  that 
more  accurate  determinations  are  secured  by  averaging  the  results 
obtained  with  the  standards  set  at  the  equivalents  of  100,  50  and  25 
mm.  of  pure  carotinoid  solutions. 

Results  by  Willstatter  and  Stall's  method.  Table  19  shows  some 
of  the  results  obtained  by  Willstatter  and  Stoll  using  their  own 
method.  The  data  are  averages  of  duplicate  determinations  reported 
in  full  by  these  investigators  and  show  the  difference  between  the 
carotin  and  xanthophyll  content  of  leaves  exposed  to  the  light  and 
those  which  are  heavily  shaded,  both  being  obtained  from  the  same 
plant.  The  fresh  leaves  which  were  in  the  shadow  were  in  some  cases 
appreciably  lower  in  carotinoids  than  the  leaves  exposed  to  the  light, 
but  this  difference  appears  to  be  due,  in  part,  to  a  higher  moisture 
content  in  the  fresh  shaded  leaves. 

The  quantitative  results  of  Willstatter  and  Stoll  show  a  very  dif- 
ferent proportion  between  carotin  and  xanthophylls  than  was  ob- 
tained by  Monteverde  and  Lubimenko,  which  can  not  be  due  entirely 
to  the  fact  that  different  plants  were  used  in  the  two  studies.    The 


QUANTITATIVE  ESTIMATION  OF  CAROTINOIDS     255 


Table  19. 


Carotin  and  Xanthophyll  Content  of  Leiaves  (Method  of 

WiLLSTATTER   AND   StOLl) 


Pigment  in 

Pigment  in 

fresh  leaves 

dry  leaves 

Xantho- 

Xantho- 

Name of  plant 

Condition 

Carotin 

phyll 

Carotin 

phyll 

mg.  per 

mg.  per 

mg.per 

mg.  per 

100  gms. 

100  gms. 

100  gms. 

100  gms. 

Samhucus  nigra 

Light-exposed 

14.1 

26.3 

52.5 

97.7 

Satnhucus  nigra 

Shaded 

6.3 

19.2 

38.5 

118.0 

Aesculus  hippocastanum 

Light-exposed 

29.3 

45.1 

79.0 

121.0 

Aesculus  hippocastanum 

Shaded 

9.3 

27.9 

37.0 

111.0 

Plataniis  acerifolia 

Light-exposed 

12.9 

27.8 

38.0 

82.5 

Platanus  acerifolia 

Shaded 

12.7 

31.1 

51.0 

125.0 

Fagus  silvatica 

Light-exposed 

18.5 

30.0 

.... 

Fagus  silvatica 

Shaded 

13.1 

25.2 

35.0 

'esio 

Populus  canadensis 

Light-exposed 

9.7 

.... 

29.0 

writer  is  inclined 'to  believe  that  the  ratio  of  1.5  to  2  molecules  of 
xanthophyll  to  1  of  carotin,  as  found  by  Willstatter  and  Stoll,  repre- 
sents more  nearly  the  true  proportion  between  the  two  classes  of 
carotinoids  as  they  exist  in  green  leaves. 

Elizabeth  Goerrig  (1917)  has  applied  the  general  principles  of  the 
Willstatter  and  Stoll  method  to  the  determination  of  the  carotin  and 
xanthophyll  content  of  yellow  autumn  leaves.  This  work  has  already 
been  discussed  in  Chapter  II  in  connection  with  the  pigments  of 
autumn  leaves,  but  it  might  be  well  to  mention  here  Miss  Goerrig's 
experience  in  applying  the  method.  She  varied  the  procedure  in  sev- 
eral particulars,  one  of  the  most  important  of  which,  as  far  as  its 
possible  effect  on  her  results  is  concerned,  was  the  preliminary  drying 
of  the  leaves  at  40°  C.,  instead  of  using  the  fresh  leaves  as  recom- 
mended in  the  original  method.  Miss  Goerrig  admits  that  the  dried 
leaves  were  difficult  to  grind  with  the  extraction  solvent  and,  in  fact, 
states  that  the  yellow  leaves  usually  retained  a  part  of  the  color  which 
could  not  be  extracted  by  the  method  recommended.  Moreover, 
the  calculation  of  the  carotin  content  of  some  of  the  leaves  using  Miss 
Goerrig's  data,  which  are  expressed  as  colorimeter  readings  only,  gives 
results  much  lower  than  Willstatter  and  Stoll  reported  for  leaves  from 
the  same  species  of  plant.  Another  important  particular  in  which 
Miss  Goerrig  modified  the  Willstatter  procedure  was  the  omission  of 
the  preliminary  extraction  with  30  per  cent  acetone  and  the  use  of 
85  to  90  per  cent  acetone  for  the  extraction  of  the  pigments  instead  of 
the  pure  acetone  recommended.  Finally,  Miss  Goerrig  used  a  0.4  per 
cent  K^CtzOj  solution  as  a  standard  in  place  of  a  0.2  per  cent  solu- 
tion.   By  setting  the  standard  at  50  mm.  a  greater  range  of  color  in- 


256  CAROTINOIDS  AND  RELATED  PIGMENTS 

tensity  was  secured  for  the  unknown  color  solutions.  No  attempt 
was  made  to  calculate  the  results  in  terms  of  carotin  and  xantho- 
phyll  content  of  the  leaves   studied. 

Miss  Goerrig  mentions  one  or  two  points  of  interest  in  connection 
with  the  remaining  steps  of  the  method,  which  were  followed  closely. 
In  the  removal  of  the  chlorophyll  by  saponification  the  alkali-chloro- 
phyllines  did  not  retain  the  xanthophylls  as  mentioned  by  Willstatter. 
Again,  in  the  final  removal  of  the  xanthophyll  to  ether  before  making 
up  the  solutions  for  the  colorimetric  reading.  Miss  Goerrig  encoun- 
tered the  most  difficult  part  of  the  whole  method.  Contrary  to  the 
statement  of  Willstatter  and  Stoll,  she  found  it  impossible  to  transfer 
all  the  xanthophylls  to  ether  by  the  slow  addition  of  water. 

Estimation  of  Fucoxanthin 

The  fucoxanthin  content  of  brown  algse  can  be  determined  by  a  col- 
orimetric method  devised  by  Willstatter  and  Page  (1914).  The  de- 
tails of  the  isolation  of  the  pigment  and  its  quantitative  estimation 
are  given  by  these  investigators  as  follows. 

The  algse  are  pressed  dry  between  filter  papers  and  ground  to  a 
fine  meal.  Except  for  Laminaria,  for  which  a  different  treatment  is 
recommended,  40  grams  of  the  meal  are  mixed  with  200  grams  of 
sand  and  macerated  with  50  cc.  of  40  per  cent  acetone,  then  twice 
with  50  cc.  of  30  per  cent  acetone.  These  extracts  are  discarded. 
The  pigments  are  then  extracted  with  pure  acetone.  Laminaria  are 
first  cut  up  into  small  pieces  and  extracted  with  30  per  cent  acetone  in 
a  beaker.  The  pulp  is  then  ground  in  a  meat  chopper  and  a  weighed 
quantity  mixed  with  sand  and  extracted  with  95  per  cent  acetone 
and  finally  with  anhydrous  acetone  until  all  the  pigments  are  ex- 
tracted. 

In  all  cases  the  pigments  are  transferred  to  ether  by  adding  300  cc. 
of  ether  to  the  acetone  solution  and  then  adding  distilled  water.  The 
ether  is  freed  from  acetone  by  very  careful  washing  with  distilled 
water  and,  after  mixing  with  an  equal  volume  of  petroleum  ether,  is 
ready  for  the  extraction  of  the  fucoxanthin.  This  is  accomplished 
by  shaking  four  times  with  an  equal  volume  of  70  per  cent  methyl 
alcohol  which  has  been  saturated  with  petroleum  ether,  the  volume 
of  the  upper  layer  being  kept  constant  by  additions  of  ether  after 
each  extraction.  The  combined  alcohol  extracts  are  freed  from  some 
xanthophyll  which  is  extracted  along  with  the  fucoxanthin  by  shaking 


QUANTITATIVE  ESTIMATION  OF  CAROTINOIDS      257 

with  an  equal  volume  of  a  mixture  of  five  parts  petroleum  ether  and 
one  part  ether.  Some  fucoxanthin  is  lost  in  this  extract  but  it  is  re- 
covered by  concentrating  the  extract  to  250  cc.  in  vacuum,  adding  an 
equal  volume  of  ether  and  extracting  twice  with  500  cc.  of  70  per 
cent  alcohol,  which  has  been  saturated  with  petroleum  ether.  The 
new  alcohol  extract  is  added  to  the  first  main  extract.  The  fuco- 
xanthin is  finally  transferred  to  ether,  which  is  freed  from  methyl 
alcohol  by  washing  with  water,  and  made  up  to  a  volume  of  250  cc.  in 
a  graduated  flask. 

The  solution  is  now  compared  in  a  colorimeter  with  either  a  stand- 
and  fucoxanthin  solution  (using  a  5  x  10'^  molar,  or  0.0304  per  cent 
solution)  or  the  0.2  per  cent  K2Cr207  standard  which  is  used  for  esti- 
mating carotin  and  xanthophylls.  The  standard  is  set  at  50  mm.  of 
standard  fucoxanthin  or  85  mm.  of  the  dichromate  solution,  which  is  its 
equivalent,  and  the  depth  of  the  unknown  solution  which  is  required 
to  match  the  color  determined.  If  the  height  of  the  unknown  is  hf  the 
content  of  fucoxanthin  in  1  kg.  of  fresh  algse  as  calculated  from  the 

1      50 

40  gram  sample  will  be  25  x  0.00608  x —x  T-^if  the  standard  fucoxan- 

thin  solution  is  used,  or  a  similar  result  if  the  dichromate  has  been 
used,  since  the  latter  will  be  set  at  the  equivalent  of  50  mm.  of  stand- 
ard fucoxanthin. 

Using  this  method  Willstatter  and  Page  determined  the  fucoxan- 
thin content  of  Fucus,  Dictyota  and  Laminaria  to  be,  respectively, 
169  mgs.,  250  mgs.,  and  81  mgs.  per  kg.  of  fresh  algae. 

Application  to  Other  Biological  Materials 

It  seems  obvious  that  the  colorimetric  methods  of  analysis  for  caro- 
tin, xanthophylls  and  fucoxanthin  as  worked  out  by  Willstatter  and 
his  co-workers  should  be  applicable  to  any  biological  material  con- 
taining these  pigments  if  a  suitable  method  can  be  devised  for  free- 
ing the  pigment  from  the  tissues  involved.  It  would  seem  that  the  Will- 
statter teohnic  can  be  applied  without  modification  to  plant  tissues, 
including  flowers,  fruits  and  leaves,  with  the  exception  of  the  fruits 
containing  lycopin,  for  which  no  quantitative  method  has  yet  been 
devised.  Moreover,  the  writer  is  not  aware  of  any  methods  for  separ- 
ating carotin  from  lycopin  so  that  even  a  quantitative  estimation  of 


258  CAROTINOIDS  AND  RELATED  PIGMENTS 

carotin  is  not  possible  in  fruits  in  which  these  two  carotinoids  are 
present  together. 

For  animal  tissues  and  fluids  containing  only  a  single  carotinoid  an 
extraction  with  ether  or  petroleum  ether  either  directly,  if  sufficiently 
dry,  or  after  treatment  with  alcohol,  if  much  water  is  present  or  if  the 
pigment  is  bound  to  protein,  should  yield  a  solution  which  may  be  used 
at  once  for  quantitative  estimation,  colorimetrically.  A  preliminary 
concentration  of  the  extract,  previous  to  comparing  with  the  standard, 
may  be  advisable.  For  blood  work  the  writer  concentrates  the  extracts 
to  the  original  volumes  of  blood  taken  (usually  10  cc.)  so  that  the 
colorimeter  readings  can  be  calculated  directly  to  the  percentage  of 
carotinoid  in  the  blood.  In  the  case  of  animal  fats,  like  butter  fat  or 
adipose  tissue  fat,  the  approximate  concentration  of  carotinoid  present 
(assuming  that  only  one  is  present)  can  be  determined  at  once  by  com- 
paring the  rendered,  melted  fat  with  the  standard  in  the  colorimeter. 
If  the  fat  is  highly  colored  the  necessity  of  keeping  the  fat  melted  can 
be  avoided  by  diluting  with  an  equal  volume  of  ether,  inasmuch  as  no 
great  difficulties  in  the  calculation  of  the  results  are  thereby  introduced. 

For  animal  tissues  and  fluids  containing  both  types  of  carotinoids 
in  sufficient  quantities  so  that  the  assumption  of  only  a  single  type  in- 
volves too  great  an  error,  it  is  not  likely  that  a  saponification  of  the 
extracts  can  be  avoided  because  of  the  presence  of  more  or  less  fat  in 
nearly  all  animal  tissues  containing  the  chromolipoids.  In  carrying 
out  this  saponification  and  subsequent  recovery  of  the  pigments  in  the 
unsaponifiable  matter,  care  should  be  taken  to  avoid  the  production  of 
aldehyde  resins  pigments  which  might  be  caused  by  the  use  of  impure 
alcohol.  One  to  two  cc.  of  10  to  20  per  cent  alcoholic  potash  for  each 
gram  of  material  extracted  for  the  pigment  analysis  would  insure  a 
large  excess  of  alkali  for  the  saponification  and  would  keep  the  volume 
of  fluids  within  the  realm  of  easily  conducted  analyses.  Following  the 
saponification  and  extraction  of  the  fat-free  pigments  from  the  soap, 
the  combined  pigments  must  be  washed  free  from  alkali  and  then  con- 
centrated to  the  lowest  possible  volume,  preferably  in  vacuum,  then 
diluted  with  petroleum  ether  of  low  boiling  point  and  the  pigments  sub- 
mitted to  fractionation  by  the  phase  test  between  the  petroleum  ether 
and  80-90  per  cent  alcohol,  preferably  methyl  alcohol.  The  separated 
carotin  and  xanthophylls  can  then  be  compared  with  the  standard  in  the 
colorimeter,  after  diluting  or  concentrating  to  a  suitable  volume.  In 
the  case  of  xanthophyll  pigments,  it  is  well  to  first  transfer  to  ether,  as 
in  the  Willstatter  technic  for  plant  tissues. 


QUANTITATIVE  ESTIMATION  OF  CAROTINOIDS     25^ 

Certain  animal  tissues  whose  carotinoid  content  may  be  desired  may- 
contain  pigments  soluble  in  alcohol  or  ether  whose  presence  would  in- 
terfere with  the  direct  analysis  of  the  extracts.  Tissues  such  as  liver, 
spleen,  kidney,  heart,  etc.  fall  in  this  class.  In  most  cases  the  foreign 
pigments  can  be  removed  by  saponification,  but  this  must  be  conducted 
with  great  care  to  avoid  the  production  of  other  foreign  pigments  which 
may  be  extracted  from  the  soap  by  ether  and  interfere,  not  only  with 
the  analysis,  but  also  with  the  true  demonstration  of  the  presence  of 
carotinoids.  Bile  pigments,  if  present,  can  usually  be  removed  by 
treating  the  fresh  tissues  with  lime  water,  previous  to  the  extraction 
with  ether,  in  order  to  form  ether-insoluble  calcium  salts.  It  must  be 
admitted,  however,  that  the  quantitative  analysis  of  tissues  of  this 
character  for  carotinoids  requires  considerable  study  before  it  can  be 
concluded  that  the  method  proposed  is  entirely  free  from  error. 

The  final  colorimetric  comparison  with  the  standard  hardly  needs 
further  comment.  It  is  obvious  that  the  0.2  per  cent  KaCraO^  solution, 
is  the  most  convenient  to  use.  For  animal  tissues,  and  perhaps  some 
plant  tissues,  the  amount  of  pigment  present  may  be  so  low  that  a  con- 
venient quantity  of  tissue  will  not  yield  sufficient  pigment  to  match  the 
dichromate  standard  at  any  of  the  equivalent  carotinoid  depths  given 
by  Willstatter  and  Stoll.  It  is  convenient  in  these  cases  to  set  the 
unknown  solution  at  a  given  depth  of  say  50  mm.  or  100  mm.  and 
match  its  color  with  the  standard  dichromate.  The  question  then  arises 
as  to  the  carotin  or  xanthophyll  equivalent  of  the  dichromate  depth 
found  in  such  an  analysis.  For  this  purpose  the  writer  has  constructed 
the  curves  shown  in  Chart  1.  These  curves  are  based  on  the  somewhat 
meager  data  given  by  Willstatter  and  Stoll  for  the  comparative  color  of 
5  X  10"^  molar  carotin  and  xanthophyll  solutions  with  the  standard 
dichromate  solution  and  also  the  comparative  color  of  the  two  pig- 
ment solutions. 

The  method  of  using  these  curves  involves  no  difficulties,  but  for  the 
sake  of  clearness  one  or  two  examples  may  be  given. 

Example  1.  A  25  mm.  layer  of  melted  butter  fat  from  cows  on  fresh 
pasture  grass  was  found  to  require  36.9  mm.  of  0.2  per  cent  KgCrgOy  in 
the  Kober  colorimeter.  Referring  to  the  carotin  curve  in  Chart  1  it 
is  seen  that  36.9  mm.  of  standard  dichromate  equals  45.2  mm.  of 
0.00268  per  cent  carotin  solution. 

Therefore,  0.00268  :  x  =r  25  :  45.2 

X  =  0.00485  per  cent  carotin  in  the  butter  fat 
(ignoring  the  sp.  g.  of  the  fat) . 


260 


CAROTIN OID&  AND  RELATED  PIGMENTS 


Example  2.  The  extract  of  10  cc.  of  blood  serum  in  10  cc.  volume 
in  ether  was  compared  colorimetrically  with  standard  dichromate.  A 
50  mm.  layer  of  serum  extract  was  found  to  equal  15  mm.  of  0.2  per 
cent  KaCraOy.    The  pigment  eventually  proved  to  be  entirely  xantho- 


10  20  30         40         SO         60         70         QO         90         lOO 

CAROTIN  OR  XANTHOPHYLL 

Quantitative  relations  between  0.2  per  cent  K2Cr207  solutions  and  5  x  10-"  molar 
solutions  of  carotin  and  xanthophyll. 


phyll.  Referring  to  the  xanthophyll  curve  in  Chart  1  it  is  seen  that 
15  mm.  of  dichromate  equals  30.5  mm.  of  0.00284  per  cent  xanthophyll 
solution. 

Therefore,  0.00284  :  x  =  50  :  30.5 

X  =  0.00173  per  cent  xanthophyll  in  the  serum 
(ignoring  the  sp.  g.  of  the  serum). 


QUANTITATIVE  ESTIMATION  OF  CAROTINOIDS     261 

Summary 

The  quantitative  estimation  of  carotinoids  in  plant  and  animal  tis- 
sues must  be  carried  out  by  colorimetric  methods.  Standard  0.2  per 
cent  potassium  dichromate  solution  serves  for  the  color  comparison 
with  carotin,  xanthophyll  and  fucoxanthin  solutions.  The  quantitative 
relations  between  the  standard  and  the  pigments  mentioned  are  de- 
scribed and  charted  in  this  chapter.  The  methods  are  also  described  in 
detail  for  preparing  plant  and  animal  tissues  for  the  analysis  as  well 
as  the  separation  of  the  individual  carotinoids  mentioned  when  present 
together  in  the  plant  extracts.  Data  are  given  showing  the  results  of 
applying  the  methods  to  various  plant  and  animal  tissues. 

No  method  has  yet  been  devised  for  estimating  lycopin  quantita- 
tively. A  suitable  standard  must  first  be  found  and  a  method  discov- 
ered for  separating  lycopin  from  carotin. 


Chapter  XI 
Function  of  Carotinoids  in  Plants  and  Animals 

The  significance  of  the  chromolipoids  in  the  metabolism  of  the  liv- 
ing organisms  in  which  they  are  found  has  not  been  discovered.  Their 
almost  universal  occurrence  in  vegetative  organisms  and  their  very 
frequent  appearance  in  animals  naturally  leads  to  the  belief  that  they 
perform  some  function  in  the  economy  of  life.  It  is  natural  for  the 
biochemist  to  seek  for  the  basis  of  the  occurrence  of  any  wide-spread 
substance  or  group  of  substances  in  living  matter  but  so  far  as  the  caro- 
tinoids are  concerned  their  significance  and  possible  functions  have  not 
got  beyond  the  realms  of  speculation.  In  presenting  the  theories  which 
have  been  advanced  it  seems  best  to  consider  the  plant  side  of  the  ques- 
tion separately  from  that  of  the  animal.  There  are  several  reasons  for 
this.  In  the  first  place  the  carotinoids  have  their  origin  in  the  plants 
and  occur  in  animals  only  as  they  are  present  in  the  food.  In  fact,  as 
already  pointed  out  in  a  previous  chapter,  the  ability  to  synthesize  the 
carotinoids  may  well  serve  as  one  of  the  means  of  distinguishing  plants 
from  animals.  In  the  second  place  the  question  has  recently  been 
raised  as  to  whether  the  carotinoid  pigments  are  identical  with  vitamin 
A,  or  related  to  these  unknowns  which  play  so  important  a  part  in  the 
nutrition  of  animals.  This  question  is  properly  considered  in  connec- 
tion with  the  possible  function  of  the  carotinoids  in  animal  life. 
Finally,  the  occurrence  of  carotinoids  in  certain  species  of  animals, 
such  as  fowls  and  cattle,  has  come  to  have  a  practical  significance  in 
connection  with  the  use  to  which  man  has  put  these  animals  in  the 
production  of  eggs  and  milk. 

Before  discussing  these  questions,  some  of  which  have  a  practical 
as  well  as  a  biochemical  point  of  view,  it  is  not  altogether  unreasonable 
to  ask  why  it  is  necessary  to  consider  that  the  carotinoids  must  play 
a  role  in  metabolism  merely  because  they  are  of  wide-spread  occur- 
rence. It  is  not  a  new  idea  that  the  lipochromes  in  animals  are  of  the 
nature  of  waste  products  of  the  organism.  Although  this  idea  was 
advanced  while  the  belief  was  generally  held  that  animals  synthesize 

262 


I 


FUNCTION  OF  CAROTINOIDS  IN  PLANTS,  ANIMALS    263 

their  own  lipochromes,  the  fact  that  animals  merely  derive  their  lipo- 
chromes  preformed  from  their  food  does  not  invalidate  the  idea  that 
these  pigments  are  merely  casual  products  in  the  animal  organism  and 
even  suggests  that  they  perform  no  useful  purpose  in  the  plants  which 
synthesize  them. 

Possible  Function  in  Plants 

Various  theories  have  been  advanced  to  explain  the  significance  of 
the  carotinoids  in  plants.  When  Arnaud  (1885)  discovered  the  pres- 
ence of  carotin  in  green  leaves  he  raised  the  question  as  to  its  possible 
relation  to  chlorophyll  and  later  (1889)  suggested  that  the  pigment 
might  play  a  role  in  plants  similar  to  that  of  the  hemoglobin  of  the 
blood.  He  also  attached  considerable  significance  to  the  fact  that 
carotin,  with  its  great  affinity  for  oxygen  when  released  from  the  living 
plant  tissues,  remains  apparently  unaltered  at  an  almost  constant  level 
when  in  the  living  leaf.  Arnaud  could  not  explain  this  except  on  the 
basis  that  the  carotin  was  constantly  undergoing  an  alternating  oxida- 
tion and  reduction  analogous  to  that  of  hemoglobin  in  the  blood. 

Zopf  looked  upon  the  lipochromes  as  reserve  products,  but  Miss  New- 
bigin  (1898)  has  aptly  stated  that  it  is  safer  to  admit  merely  that  they 
often  occur  in  association  with  reserves.  Zopf,  however,  apparently 
limited  his  conception  of  carotin  as  a  reserve  substance  to  certain  fungi, 
such  as  the  Uridinece  (rusts)  and  certain  molds.  The  idea  was  based 
upon  his  observation  that  the  pigment  seems  to  concentrate  in  the 
spores  of  these  plants  and  later  to  disappear  during  germination. 
Kohl  (1902J)  accepted  this  idea  and  in  addition  stated  that  he  believed 
that  carotin  acted  as  a  reserve  substance  in  the  carrot  root. 

Kohl  has,  in  fact,  given  us  the  most  comprehensive  conception  of  the 
various  roles  which  carotin  may  play  in  plant  life.  Primarily  he  be- 
lieves with  Engelmann  (1887)  that  carotin  shares  with  chlorophyll 
the  work  of  carbon  dioxide  assimilation,  and  that  this  lies  chiefly  in 
its  energetic  absorption  of  a  large  part  of  the  blue-violet  rays  of  sun- 
light. This  light  is  transformed  into  heat,  a  property  which  Stahl 
(1896)  believed  anthocyanin  and  carotin  shared,  permitting  the  pig- 
ment to  act  indirectly  as  a  catalyst  for  various  metabolic  processes, 
including  the  decomposition  of  the  atmospheric  carbon  dioxide.  There 
can  be  no  doubt  that  the  spectroscopic  properties  of  the  carotinoids  are 
one  of  the  strongest  arguments  in  favor  of  the  view  that  they  perform 
some  definite  function  in  the  plant.  Whether  the  light  absorbed  is 
transformed  as  Kohl  believes  or  whether  it  serves  some  other  purpose 


264  CAROTINOIDS  AND  RELATED  PIGMENTS 

is  difficult  to  determine.  Kohl  (1906b)  offered  as  proof  of  his  theory 
the  fact  that  etiolated  leaves  which  would  not  turn  green  in  partial 
vacuum  did  so  when  the  partial  vacuum  was  replaced  by  oxygen-free 
COg.  He  believed  that  this  experiment  shows  that  the  carotin  in  the 
etiolated  leaf  is  able  to  transform  the  COg  into  oxygen  for  the  forma- 
tion of  chlorophyll. 

The  spectroscopic  absorption  properties  of  the  carotinoids  may  serve 
the  purpose  of  protecting  the  cell  enzymes  against  the  destructive  ac- 
tion of  certain  light  rays,  according  to  Went  (1904).  This  theory 
has  been  supported  by  Kohl  (1906c)  also,  who  states  that  he  was  able 
to  establish  experimentally  that  carotin  solutions  exert  a  protective 
action  towards  diastase  through  their  absorption  of  the  violet  and 
ultra-violet  rays  beginning  about  420t.i[x. 

With  reference  to  carotin  acting  in  a  respiratory  role  through  its 
power  to  absorb  oxygen,  as  suggested  by  Arnaud,  Kohl  (1900j)  thinks 
that  it  may  so  act  in  the  chloroplastids,  but  that  no  general  respiratory- 
role  can  be  ascribed  to  it  inasmuch  as  respiration  is  known  to  proceed 
just  as  normally  in  colorless  cells  as  in  those  which  are  pigmented. 
From  a  biological  point  of  view  Kohl  believes  that  carotin  shares 
with  other  pigments  of  flowers  and  fruits  the  function  of  an  effective 
lure  for  insects  and  birds  and  other  animals,  in  connection  with  the 
spreading  of  seeds  and  pollen. 

Willstatter  and  Mieg  (1907)  agree  with  Arnaud  that  the  most  likely 
functions  of  the  carotinoids  are  related  to  their  great  affinity  for  oxy- 
gen. They  are  inclined  to  regard  the  work  of  these  pigments  in  the 
light  of  oxygen  transference,  however,  rather  than  as  directly  con- 
cerned with  the  oxygen  assimilation.  They  accept  the  possibility  of  a 
certain  amount  of  oxygen  absorption.  They  are  careful  to  point  out, 
however,  that  xanthophyll  cannot  well  be  the  end  product  of  this 
oxygen  absorption;  but  that  this  product  is  probably  concerned  with 
the  regulation  of  the  oxygen  pressure  in  the  plant  cells.  With  refer- 
ence to  a  possible  part  which  the  carotinoids  might  play  in  carbon 
dioxide  assimilation  Willstatter  and  Stoll  express  the  belief  that  an 
experimental  demonstration  is  needed  of  the  possibility  of  the  caroti- 
noids acting  in  such  a  role  alone,  inasmuch  as  it  is  a  chemical  function 
difficult  to  understand  for  these  pigments.  It  would  appear  that  Kohl 
(1906c)  has  already  furnished  a  certain  amount  of  evidence  along  this 
line,  but  this  does  not  seem  to  be  generally  accepted  by  the  plant 
physiologists.  In  fact,  Miss  Irving  (1910)  has  shown  that  the  green- 
ing of  etiolated  shoots  is  not  indicative  of  the  power  of  carbon  assimila- 


FUNCTION  OF  CAROTINOIDS  IN  PLANTS,  ANIMALS     265 

tion,  but  that  this  ability  is  still  lacking  even  in  such  shoots  which 
have  developed  a  considerable  amount  of  green  color. 

A  somewhat  different  aspect  to  the  possible  function  of  the  caroti- 
noids  in  the  chloroplastids  is  given  by  another  theory  of  Willstatter 
and  Stoll  in  which  it  is  supposed  that  the  carbon  dioxide  assimilation 
is  controlled  by  the  equilibrium  between  the  chlorophyll  components 
a  and  b,  and  that  this  equilibrium  is  in  turn  controlled  by  the  caroti- 
noids.  The  process,  as  imagined  by  Willstatter,  is  as  follows.  Carbon 
dioxide  is  attracted  by  the  affinity  of  the  magnesium  compounds 
(chlorophylls)  for  COg,  and  is  at  once  reduced  by  chlorophyll  a. 
Chlorophyll  a  is  thereby  oxidized  to  chlorophyll  b.  Carotin  then  with- 
draws the  oxygen  from  chlorophyll  b,  reducing  it  again  to  chlorophyll 
a,  the  carotin  at  the  same  time  being  oxidized  to  xanthophyll.  The 
reduction  of  the  xanthophyll  to  carotin,  in  order  to  complete  the  cycle 
of  Willstatter's  theory,  is  effected  by  a  reductase.  One  difficulty  with 
this  theory  is  that  Willstatter  himself,  as  he  has  pointed  out,  does  not 
admit  that  carotin  can  be  oxidized  to  xanthophyll.  The  ability  of 
carotin  to  reduce  chlorophyll  b  and  thereby  become  oxidized  is  un- 
questioned, as  evidenced  by  its  strong  reducing  action  on  ferric  salts. 
Xanthophyils,  however,  share  this  property  with  carotin.  At  the  same 
time  some  support  is  given  to  the  theory  of  a  functional  relation  be- 
tween the  chlorophylls  and  carotinoids  by  the  possibility  that  fucoxan- 
thin  plays  the  part  of  chlorophyll  b  in  the  brown  algae,  which  lack 
this  chlorophyll  component. 

Ewart  has  recently  (1915)  attempted  to  show  that  carotin  and  xan- 
thophyll can  play  a  part  in  photo-synthesis.  His  experiinents  purport 
to  show  that  carotin  yields  HCHO  when  submitted  to  photo-oxidation 
in  a  stream  of  pure  oxygen  and  that  xanthophyll  yields  both  HCHO 
and  sugar  under  similar  conditions.  In  view  of  the  fact  that  Ewart's 
conception  of  xanthophyll  includes  the  idea  that  it  "is  soluble  in  water 
and  in  any  mixture  of  alcohol  and  water,"  and  also  since  there  is  no 
assurance  that  his  carotin  was  free  from  impurities,  his  results  can  not 
be  given  unqualified  acceptance.  In  the  same  paper  Ewart  claims  to 
have  produced  xanthophyll  from  chlorophyll,  but  Jorgensen  and  Kidd 
(1917)  have  shown  that  the  "xanthophyll"  which  Ewart  produced  in 
his  experiments  was  probably  phaeophytin. 

The  question  of  the  origin  of  the  carotinoids  in  plants,  which  is  sug- 
gested by  Ewart's  attempt  to  produce  xanthophyll  from  chlorophyll, 
is  closely  related  to  the  question  of  their  function.  The  fact  that  the 
carotinoids  form  in  etiolated  plants  without  chlorophyll  is  a  strong 


^66  CAROTINOIDS  AND  RELATED  PIGMENTS 

point  in  favor  of  their  independent  existence,  but  does  not,  of  course, 
show  that  the  chlorophylls  and  carotinoids  do  not  arise  from  a  com- 
mon nucleus.  From  a  chemical  point  of  view  the  most  likely  sub- 
stance which  could  thus  give  rise  to  both  carotinoids  and  chlorophyll 
would  be  isoprene,  CgHy,  the  terpene  ''baustein"  which  may  go  to 
phytol  on  the  one  hand,  as  Willstatter  believes,  and  perhaps  could  also 
go  to  carotin  on  the  other. 

Very  little  study  has  been  given  to  the  physiological  conditions  which 
govern  the  formation  of  the  carotinoids  in  plants  or  to  the  problem  of 
the  relations  between  the  different  carotinoids  or  between  the  caroti- 
noids and  other  plant  constituents.  The  Toblers  (1912)  observed  that 
the  carotin  content  of  carrots  increased  during  the  formation  of  starch 
from  sugar,  but  it  is  difficult  to  decide  from  this  observation  that  the 
carotin  plays  any  part  in  the  process.  These  investigators  (1910b, 
1912)  have  also  shown  that  the  formation  of  carotin  and  lycopin  in  the 
ripening  of  tomato  fruits  is  coincident  with  the  destruction  of  chloro- 
phyll. Lubimenko  (1914a)  has  concluded  that  the  lycopin  forms  in 
this  case  at  the  expense  of  the  chlorophyll,  but  there  is  no  chemical 
basis  for  assuming  that  this  indicates  that  the  carotinoids  are  actually 
formed  from  the  chlorophyll. 

Duggar  (1913)  made  an  especially  interesting  study  of  the  develop- 
ment of  carotinoids  in  tomato  fruits.  He  found  that  the  factor  for 
carotin  formation  and  the  factor  for  lycopersicin  (lycopin)  formation 
are  present  together  in  the  fruits  which  normally  redden  on  ripening, 
but  that  the  formation  of  the  red  carotinoid  could  be  partially  or  com- 
pletely suppressed  by  ripening  the  green  fruits  at  a  temperature  of 
30°  C.  or  above.  At  these  temperatures  the  fruits  ripened  with  a  yel- 
low color  and  contained  only  carotin  and  xanthophylls.  The  inhibition 
of  the  lycopin  was  found  to  be  proportional  to  the  temperature  (be- 
tween 30°  and  37°  C),  but  was  inversely  related  to  the  age  of  the 
fruits,  the  oldest  fruits  requiring  a  higher  temperature  for  the  sup- 
pression of  reddening.  The  failure  of  the  fruits  to  develop  lycopersicin 
at  the  higher  temperatures  was  found  to  be  a  true  suppression,  inas- 
much as  the  red  pigment  formed  rapidly  when  the  yellow  fruits  were 
returned  to  a  lower  temperature.  Duggar  was  also  able  to  show  very 
satisfactorily  both  by  means  of  the  microscope  and  the  spectroscope 
that  the  lycopin  which  formed  in  these  cases  could  not  have  been  de- 
rived from  the  carotin  present. 

Duggar  also  made  a  study  of  other  factors  entering  into  the  forma- 
tion of  the  lycopin  of  tomatoes,  with  the  result  that  the  synthesis  of 


FUNCTION  OF  CAROTINOIDS  IN  PLANTS,  ANIMALS    267 

the  pigment  was  found  to  be  independent  of  light,  but  dependent  upon 
oxygen.  The  fruits  failed  to  redden  in  all  cases  of  ox;^'gen  exclusion 
even  at  a  favorable  temperature,  but  assumed  a  greenish  yellow,  yellow 
or  yellow-orange  color  with  an  accompanying  loss  of  chlorophyll. 
Whether  the  latter  colors  were  due  to  a  formation  of  carotin  and  xan- 
thophyll  in  the  atmospheres  of  hydrogen  and  nitrogen  employed,  Or 
whether  the  carotinoids  were  merely  revealed  by  the  destruction  of  the 
chlorophyll  is  not  clear.  Observations  were  also  made  on  the  catalase 
activity  of  the  fruits  as  well  as  their  titratable  acidity  under  the  con- 
ditions of  lycopin  suppression,  with  the  result  that  it  was  found  that  a 
very  low  catalase  activity  and  decreased  acidity  accompany  the  con- 
ditions which  suppress  the  formation  of  the  pigment.  It  is  apparent, 
however,  that  these  are  not  the  only  factors  concerned. 

Possible  Function  in  Animals 

A  quarter  of  a  century  ago  the  majority  of  the  biologists  accepted 
the  idea  that  all  the  visible  pigments  of  animals,  including  the  lipo- 
chromes,  are  essential  products  of  the  animal  metabolism.  The  pre- 
vailing theories  of  evolution  looked  upon  animal  colorations  as  factors 
in  the  existence  of  the  species  which  had  persisted  solely  for  some 
useful  purpose.  The  theories  which  described  the  function  of  the 
pigments  in  various'  terms,  such  as  Protective  Coloration,  Warning 
Coloration,  Mimicry,  Sexual  Attraction,  etc.,  all  had  their  followers. 
it  is  not  our  purpose  to  discuss  these  theories.  One  can  find  them 
both  adequately  defended  and  impartially  criticized  in  the  treatises 
current  during  the  closing  years  of  the  past  century.  Suffice  it  to  say 
that  the  writer  does  not  possess  a  biological  viewpoint  which  is  sufii- 
ciently  developed  along  academic  lines  to  appreciate  "function"  as  an 
abstract  attribute  of  living  organisms.  Function,  to  be  real,  according 
to  his  conception,  must  be  concrete  or  physiological.  •  Perhaps  there 
are  those  who  will  regard  this  as  going  from  one  extreme  to  the  other. 
If  so,  the  explanation  lies  in  the  fact  that  this  monograph  deals  only 
with  animal  pigments  which  are  derived  from  the  food.  Such  pig- 
ments, if  possessing  a  function,  must  be  linked  with  the  physiological, 
in  this  case  the  nutritional  or  metabolic  processes  of  the  body. 

We  have  seen  in  Chapter  VII  that  great  variation  exists  among  .dif- 
ferent species  of  mammals  with  respect  to  their  ability  to  absorb  the 
carotinoids  from  their  food  and  deposit  the  pigments  in  their  tissues, 
or,  if  one  prefers  the  opposite  point  of  view,  to  destroy  the  carotinoids 


268  CAROTINOIDS  AND  RELATED  PIGMENTS 

ingested  and  thus  prevent  their  deposition  in  the  tissues.  This  fact 
alone  seems  to  argue  against  the  carotinoids  performing  any  general 
physiological  function  in  animals ;  a  priori,  a  substance  of  general  value 
in  nutrition  would  most  likely  have  a  general  occurrence  and  would  at 
least  always  be  present  in  the  animal  body.  The  complete  absence 
of  carotinoids  from  the  tissues  and  secretions  of  certain  species  of  ani- 
mals and  their  almost  complete  absence  from  others,  even  on  diets  rich 
in  carotinoid  pigments,  furnishes  a  sufficient  basis,  at  least  from  a  teleo- 
logical  standpoint,  for  rejecting  any  theory  that  the  carotinoids  exert 
a  physiological  function  in  animal  life. 

The  writer  became  interested  in  this  question  from  an  experimental 
point  of  view  in  1912,  when  it  was  found,  in  connection  with  the  bio- 
logical origin  of  the  lipochromes  of  cattle,  that  the  new-born  calf  of  a 
highly  pigmented  breed  of  cattle  showed  an  almost  Complete  absence 
of  carotinoids.  This  suggested  the  idea  of  raising  animals  to  maturity 
on  carotinoid-free  diets,  particularly  those  species  which  normally  de- 
posit the  carotinoids  in  their  tissues.  Later,  after  the  writer  (1915) 
had  shown  that  the  lipochromes  of  fowls  are  derived  from  plant  xantho- 
phyll,  plans  were  laid  for  carrying  out  such  an  experiment  on  these 
animals,  because  of  the  obvious  advantages  associated  with  a  smaller, 
more  rapidly  growing  species. 

The  problem  was  primarily  one  of  selecting  a  ration  entirely  devoid 
of  carotinoids,  particularly  xanthophyll,  but  otherwise  adequate  for 
normal  growth.  The  problem  had  the  added  interest  that  the  rapidly 
growing  subject  of  vitamins  had  already  (1916)  indicated  a  casual  re- 
lationship between  the  occurrence  of  fat-soluble  vitamin  A  and  caroti- 
noids in  certain  foods  such  as  butter  and  green  leaves,  and  the  absence 
of  both  substances  from  lard.  The  experiments  showing  the  possi- 
bility of  raising  fowls  on  diets  lacking  the  natural  pigment  of  their 
adipose  tissue,  which  were  begun  in  1916  and  were  reported  by  Palmer 
and  Kempster  (1919a)  were  not  designed,  however,  to  show  the  rela- 
tion between  carotinoids  and  vitamin  A.  The  writer  dismissed  the 
possibility  of  any  such  relation  as  the  result  of  the  experiment  carried 
out  in  the  winter  of  1916-17  in  which  young  chickens  weighing  700 
to  750  grams  were  raised  to  maturity  and  exhibited  normal  fecundity 
on  carotinoid-free  rations.  The  successful  termination  of  a  later  ex- 
periment in  this  series,  in  which  a  flock  of  50  chickens  was  raised  from 
hatching  to  maturity  on  similar  diets,  showed  conclusively  for  the 
first  time  that  a  species  of  animal  which  is  normally  pigmented  with 


FUNCTION  OF  CAROTINOIDS  IN  PLANTS,  ANIMALS    269 

carotinoids  does  not  require  these  pigments  for  its  growth  or  for  the 
reproduction  of  its  kind. 

While  the  last  experiment  was  in  progress  Drummond  (1919)  re- 
ported the  failure  of  pure  crystalline  carotin,  fed  at  the  rate  of  0.003 
per  cent  of  the  ration  to  improve  the  condition  of  albino  rats  suffering 
from  vitamin  A  deficiency ;  while  Steenbock,  Boutwell  and  Kent  (1919) , 
on  the  other  hand,  were  calling  attention  to  certain  new  associations  of 
yellow  pigmentation  and  vitamin  A  and  were  suggesting  that  the  two 
were  at  least  associated  in  some  way.  Although  the  statement  was 
made  that  vitamin  A  "is  not  carotin,"  this  was  later  retracted  by  Steen- 
bock (1919)  and  the  provisional  assumption  advanced  that  this  vita- 
min is  one  of  the  carotinoid  pigments.  In  support  of  this  assumption 
Steenbock  and  his  associates  have  published  a  series  of  papers  showing 
that  a  rather  close  correlation  exists  between  carotinoid  pigmentation 
and  vitamin  A  content  of  roots  (Steenbock  and  Gross,  1919;  Steen- 
bock and  Sell,  1922),  maize  (Steenbock  and  Boutwell,  1919a),  leaves 
(Steenbock  and  Gross,  1920) ,  and  peas  (Steenbock,  Sell  and  Boutwell, 
1921),  as  determined  by  feeding  pigmented  and  colorless  varieties  of 
these  plant  products  to  albino  rats.  However,  in  studying  the  extract- 
ability  of  vitamin  A  from  carrots,  alfalfa,  and  yellow  maize  by  fat 
solvents,  Steenbock  and  Boutweli's  (1920b)  results  show  that  highly 
colored  extracts  ^  do  not  exhibit  the  vitamin  activity  which  would  be 
expected  if  vitamin  A  is  a  carotinoid;  and  yellow  maize,  even  after 
extraction  with  hot  ether  is  shown  to  have  lost  very  little  vitamin  A, 
although  there  must  have  been  a  considerable  loss  of  pigment.  On  the 
other  hand,  especially  favorable  to  Steenbock's  theory  was  the  finding 
in  this  paper  that  the  fat-soluble  vitamin  follows  the  carotin  in  the 
application  of  the  phase  test  to  the  unsaponifiable  extracts  from  alfalfa 
leaves.  When  one  bears  in  mind,  however,  that  the  solvents  employed 
in  the  separation  of  the  carotinoids  by  this  method  are  respectively 
exceedingly  poor  and  very  excellent  fat  solvents,  it  is  not  surprising 
that  the  fat-soluble  vitamin  will  follow  the  substance  which  goes  into 
the  better  fat  solvent. 

When  Steenbock,  Sell  and  Buell  (1921)  attempted  to  obtain  support 
for  Steenbock's  theory  among  animal  products  the  correlation  between 
pigmentation  and  vitamin  content  was  so  poor  when  comparing  prac- 
tically colorless  codliver  oil  with  butter  fats  of  high  and  low  color  that 

1  Drummond  and  Zilva  (1922)  have  substantiated  this.  They  find  that  only  "very 
slight"  growth  in  rats  is  promoted  by  as  much  as  2  grams  daily  of  the  crude  oil 
extracted  from  yellow  maize  by  petroleum  ether.  This  is  a  high  proportion  of  the 
diet   of   young    rats. 


270  CAROTINOIDS  AND  RELATED  PIGMENTS 

Steenbock  has  been  forced  to  abandon  his  position  that  the  two  sub- 
stances may  be  identical  and  to  admit  that  their  "coincident  occurrence 
in  nature  might  be  due  to  physiological  determination,  pure  and  sim- 
ple." The  attempts  to  show  some  correlation  between  the  color  of 
perinephritic  beef  fat  and  vitamin  A  content  in  the  same  paper  are  not 
especially  convincing  on  close  examination,  especially  when  the  results 
are  compared  with  butter  fat  of  like  color  fed  at  much  lower  levels. 
In  addition,  the  statement  is  made  that  egg  yolks  of  a  light  color  but 
with  a  normal  vitamin  content  can  be  produced  on  specially  selected 
rations,  which  confirms  the  observations  of  Palmer  and  Kempster 
(1919b)  and  Palmer  and  Kennedy  (1921). 

The  lack  of  correlation  between  pigmentation  and  vitamin  content  of 
animal  fats  was  first  pointed  out  by  Drummond  and  Coward^  (1920) 
for  butter  fat  and  a  large  number  of  other  fats  and  oils  (including 
vegetable  oils).  It  is  of  interest  that  colorless  dog  fat  and  colorless 
perinephritic  pig  fat  were  relatively  rich  in  vitamin  A.  Miss  Stephen- 
son (1920)  further  corroborated  this  by  decolorizing  butter  fat  with 
charcoal  without  impairing  in  any  way  its  vitamin  content.  This  ex- 
periment, however,  requires  confirmation,  primarily  because  of  the  re- 
markably small  amount  of  charcoal  which  was  used.  As  stated  in 
Chapter  IX,  the  writer  has  not  yet  succeeded  in  duplicating  these  de- 
colorizations  with  only  2.5  per  cent  of  any  decolorizing  carbon  which 
he  has  been  able  to  secure. 

Further  proof  that  vitamin  A  is  not  necessarily  associated  with 
carotinoids  was  furnished  by  Palmer  and  Kennedy  (1921)  who  found 
that  albino  rats  grew  normally  and  reproduced  on  diets  in  which  prac- 
tically carotinoid-free  ewe  milk  fat  (containing  0.00014  per  cent  caro- 
tin) furnished  the  vitamin  A  in  the  ration  at  levels  of  5  to  9  per  cent, 
and  that  similar  results  followed  the  use  of  carotinoid-free  egg  yolk 
produced  by  hens  on  diets  made  up  of  selected  white  corn,  skim  milk, 
pork  liver  (about  10  per  cent)  and  grit.  With  the  rations  containing 
carotinoids,  the  best  results  were  secured  with  only  0.126  parts 
of  carotinoid  per  million  of  ration.  This  is  very  much  less  pigment 
than  Drummond  or  Miss  Stephenson  fed  to  rats  without  success.  In 
opposition  to  this  result  Steenbock,  Sell,  Nelson  and  Buell  (1920)  have 

==  Previous  to  this,  Rosenheim  and  Drummond  (1920)  were  much  attracted  by  the 
idea  of  an  intimate  relationship  between  carotinoids  and  vitamin  A,  and  abandoned 
it  very  reluctantly  when  they  were  unable  to  establish  an  identity  of  vitamin  A  with 
either  carotin  or  xanthophyll.  Van  den  Bergh,  Muller  and  Broekmeyer  (1920)  have 
also  supported  Steenbock's  theory,  without,  however,  submitting  it  to  experimental 
verification. 


FUNCTION  OF  CAROTINOIDS  IN  PLANTS,  ANIMALS    271 

stated  that  "carotin  of  constant  melting  point  through  a  number  of 
crystallizations  was  always  found  to  induce  growth  in  rats  after  growth 
had  been  suspended  by  a  lack  of  fat-soluble  vitamin  in  the  diet."  It 
is  not  stated  how  much  carotin  was  fed.  The  statement  is  followed, 
however,  by  the  naive  assertion  that,  "in  spite  of  this  it  is  not  meant 
to  infer  that  the  fat-soluble  vitamin  is  necessarily  a  pigment."  In  the 
same  note,  Steenbock  and  his  associates  state  that  they  have  prepared 
crystalline  acetyl  derivatives  of  constituents  in  the  non-saponifiable 
vitamin  fraction  of  the  extracts  from  alfalfa  hay,  without  resultant  de- 
struction of  the  vitamin.  This  fact  alone  is  incompatible  with  a  caroti- 
noid  nature  for  vitamin  A.  These  pigments  being  hydrocarbons  or 
hydrocarbons  with  an  ether-like  nucleus  are  quite  incapable  of  forming 
acetyl  derivatives. 

Some  light  on  the  cause  of  the  coincident  occurrence  of  vitamin  A 
and  carotinoids  is  furnished  by  the  recent  experiments  of  Coward  and 
Drummond  (1921)  who  find  that  the  synthesis  of  vitamin  A  is  associ- 
ated with  the  formation  of  chlorophyll.  Their  results  showing  the  pres- 
ence of  little  if  any  fat-soluble  vitamin  in  etiolated  seedlings  and  red 
sea-weeds,  which  are  certainly  not  wanting  in  carotinoids,  but  which 
lack  chlorophyll,  support  our  own  conclusion  that  vitamin  A  and 
carotinoids  are  not  necessarily  associated.  The  finding  is  of  added 
interest  because  it  shows  that  examples  of  this  lack  of  association 
occur  in  the  vegetable  world  as  well  as  in  the  animal  kingdom. 

These  results  when  considered  together  with  the  results  of  Drum- 
mond and  Steenbock,  as  well  as  those  of  Palmer  and  Kennedy,  show- 
ing the  lack  of  definite  correlation  between  the  carotin  and  vitamin 
content  of  milk  fat,  indicate  very  clearly  that  the  animals  which  trans- 
fer carotin  abundantly  from  the  food  to  the  milk,  as  well  as  those 
which  do  not  do  so,  have  the  power  to  separate  pigment  and  vitamin. 
The  writer  suggests  that  the  presence  of  appreciable  amounts  of  vita- 
min A  in  the  almost  colorless  butter  fat  examined  by  Steenbock  may 
have  come  from  more  or  less  yellow  maize  in  the  diet  of  the  cows. 
Palmer  and  Eckles  (1914a)  found  that  yellow  maize  has  no  appreciable 
effect  on  the  color  of  butter  fat,  but  it  should  bolster  up  the  vitamin 
content  of  the  butter,  according  to  Steenbock's  findings  on  the  rela- 
tive vitamin  content  of  yellow  and  white  maize.  In  an  analogous  man- 
ner it  should  be  possible  to  produce  eggs  with  low  pigmented  yolks, 
high  in  vitamin  A,  by  limiting  the  pigmented  part  of  the  hen's  ration  to 
carrots,  for  the  writer  (1915)  has  shown  that  the  feeding  of  carrots 
has  little  influence  on  the  color  of  the  yolks  of  hen's  eggs. 


272  CAROTINOIDS  AND  RELATED  PIGMENTS 

An  association  of  carotinoids  with  other  vitamins  than  vitamin  A 
and  with  the  results  of  other  dietetic  deficiencies  has  also  been  sug- 
gested. Wiehuizen  and  others  (1919)  called  attention  to  the  low  lipo- 
chrome  content  of.  the  blood  serum  in  the  case  of  human  beriberi  and 
inferred  a  relationship  between  lipochromes  and  the  antineuritic  vita- 
min by  stating  that  animal  and  vegetable  substances  with  a  high 
lipochrome  content  also  have  a  high  anti-beriberi  value.  It  hardly 
seems  possible  that  anyone  with  a  thorough  knowledge  of  the  distribu- 
tion and  properties  of  vitamin  B  could  give  this  suggestion  any  serious 
thought. 

A  somewhat  different  conception  of  the  significance  of  carotinoids 
in  nutrition  is  presented  by  McCarrison  (1920)  who  noted  that  butter 
made  from  milk  of  cows  on  green  feed,  and  therefore  high  in  pigment, 
afforded  greater  protection  against  edema  of  the  adrenals  of  pigeons 
fed  on  autoclaved  rice  than  less  highly  colored  butter  fat  made  from 
milk  of  cows  on  dry  feed.  McCarrison  suggests  that  the  hypothetical 
anti-edema  substance  may  be  of  the  nature  of  a  lipochrome,  but  his  re- 
sults can  also  be  explained  on  the  basis  of  the  seasonal  (dietary)  varia- 
tion in  the  vitamin  A  content  of  butter. 

We  see  from  the  foregoing  discussion  that  there  is  little  evidence  to 
support  the  idea  that  the  carotinoids  exert  a  definite  physiological 
function  either  in  the  species  of  animals  in  which  they  are  visible  after 
absorption  or  in  those  animals  which  do  not  appear  to  absorb  the  pig- 
ments at  all.  Curiously  enough,  however,  a  very  practical  use  has  been 
made  of  the  appearance  of  carotinoid  pigments  in  certain  of  the  visible 
skin  parts  of  some  species  which  absorb  the  pigments.  One  may  per- 
haps be  justified  in  discussing  these  uses  briefly  in  connection  with  the 
possible  function  of  the  pigments,  although  the  function  in  these  cases 
is  in  the  service  of  man. 

Practical  poultry  men  in  this  country  have  recognized  for  a  number 
of  years  that  a  relation  exists  between  the  amount  of  yellow  pigment 
in  the  shanks,  ear  lobes,  beaks,  etc.,  of  hens  of  certain  breeds  of  poultry, 
such  as  Leghorns,  Plymouth  Rocks,  Wyandottes,  and  Rhode  Island 
Reds,  and  their  previous  egg  laying  activity.  When  Blakeslee  and 
Warner  (1915a,b)  and  Blakeslee,  Harris,  Warner  and  Kirkpatrick 
(1917)  made  extensive  biometric  analyses  of  data  collected  to  deter- 
mine the  character  and  extent  of  this  relation  it  was  found  that  a  defi- 
nite positive  correlation  existed  between  pale  shanks,  ear  lobes,  beak, 
etc.,  and  a  recent  more  or  less  large  egg  production.  As  the  result  of 
these  studies  American  poultry  experts  have  made  extensive  use  of  the 


FUNCTION  OF  CAROTINOIDS  IN  PLANTS,  ANIMALS    273 

appearance  of  the  normally  colored  skin  parts  of  these  breeds  of  poultry 
at  the  end  of  the  laying  season  for  the  purpose  of  culling  out  the  un- 
profitable hens.  This  method  of  determining  heavy  from  light  laying 
fowls  is  not  applicable,  of  course,  to  the  breeds  of  poultry,  such  as  the 
English  Orpingtons,  which  never  show  yellow  pigment  in  the  visible 
skin  parts,  although  normally  their  adipose  tissue  and  egg  yolks  are 
colored  with  xanthophyll. 

Palmer  and  Kempster  (1919b)  made  a  study  of  the  physiological 
cause  of  the  fading  of  the  visible  skin  parts  during  egg  laying.  The  as- 
certaining of  the  correct  cause  of  this  phenomenon  was  made  possible 
by  the  success  which  we  had  in  raising  a  flock  of  pigmentless  White 
Leghorn  fowls  to  maturity,  the  females  showing  normal  egg  laying  ac- 
tivity. It  was  found  that  xanthophyll  appeared  in  the  skin  of  non-lay- 
ing fowls  within  a  few  days  ^  after  feeding  xanthophyll-containing 
foods,  but  that  no  pigment  whatever  appeared  in  the  skin,  and  almost 
none  in  the  adipose  tissue  of  the  hens  which  were  laying,  although  only 
moderately  (two  eggs  or  less  a  week) ,  even  after  a  month  on  xantho- 
phyll-rich  diets.  The  blood  serum  and  egg  yolks  contained  an  abun- 
dance of  xanthophyll.  It  was  also  found  that  when  pigmented  fowls 
which  were  not  laying  (in  these  cases  cockerels  were  used)  were  placed 
on  carotinoid-free  diets,  they  gradually  lost  the  pigment  from  the  visi- 
ble skin  parts  in  the  same  manner  as  laying  hens.  Histological  studies 
of  the  skin  during  this  fading  indicated  that  the  movement  of  the  pig- 
ment was  outward  from  the  rete  of  Malphigi,  where  it  is  chiefly  local- 
ized, towards  the  epidermis.  No  evidence  was  obtained  that  the  loss 
of  pigment  was  due  to  resorption  but  the  indications  were  rather  of 
a  normal  replacement  of  epidermis  cells  by  the  columnar  pigmented 
cells  of  the  Malphigian  layer  from  beneath  which  carried  less  and  less 
pigment  because  the  supply  of  pigment  in  the  food  had  been  cut  off. 
The  fading  of  a  highly  pigmented  skin  is  very  gradual  and  usually  re- 
quires several  months  in  the  absence  of  carotinoid  from  the  food  or  in 
the  case  of  egg  laying. 

The  collective  data  were  interpreted  to  mean  that  the  fading  of  the 
skin  during  egg  laying  is  the  result  of  the  deflection  of  the  xanthophyll 
of  the  food  to  the  ovaries,  resulting  in  a  cutting  off  of  the  pigment 
which  would  otherwise  be  excreted  by  the  skin,  the  net  result  being  the 
same  as  if  the  xanthophyll  was  no  longer  being  ingested  in  the  food. 
The  writer  believes  that  a  continuous  formation  of  ova,  but  not  neces- 

'  In  one  case  the  color  was  distinctly  visible  in  72  hours  after  xanthophyll  was  intro- 
duced into  the  ration. 


274  CAROTINOIDS  AND  RELATED  PIGMENTS 

sarily  with  great  frequency,  is  required  to  prevent  the  excretion  of 
xanthophyll  by  way  of  the  skin,  and  thus  brings  about  a  gradual  fading 
of  the  visible  skin  parts.  Whether  there  is  a  mobilization  of  pigment 
in  other  organs  of  the  body,  such  as  the  liver,  was  not  determined  in 
our  experiments. 

Rosenheim  and  Drummond  ( 1920)  have  expressed  the  view  that  this 
deflection  of  xanthophyll  to  the  ovaries  during  egg  laying  indicates  that 
the  pigment  is  required  for  a  definite  and  important  function  in  the 
egg  and  that  this  fact  thus  supports  the  theory  that  the  carotinoids  are 
related  to  the  vitamins.  It  is  just  as  reasonable  to  suppose,  however, 
that  the  egg  yolk  is  an  easier  pat*h  of  excretion  for  a  fat-soluble  pig- 
ment than  is  the  skin,  just  as  the  kidneys  are  ordinarily  the  chief  path 
of  excretion  of  water-soluble  waste  products.  Nevertheless,  it  might 
be  worth  while  to  investigate  the  relation  between  this  whole  phenome- 
non and  the  more  recent  interpretation  of  the  effect  of  Nile  blue  on 
the  pigment  granules  in  the  epidermis  of  the  chicken  skin,  namely,  that 
the  pigment  is  transported  there  in  association  with  fatty  acids.  It  is 
possible  that  the  concentration  of  the  fat  synthesizing  powers  of  the 
hen  in  the  ovaries  during  egg  laying  prevents  the  secretion  of  fatty 
acids  by  the  blood  capillaries  and  thus  causes  a  concentration  of  xan- 
thophyll in  the  fat  laid  down  in  the  ova.  This  does  not  explain,  how- 
ever, why  Sudan  III,  a  fat  dye,  never  appears  in  the  skin  when  fed  to 
either  laying  or  non-laying  fowls,  although  it  appears  abundantly  in 
the  egg  yolk,  bone  marrow  and  adipose  tissue,  and  feathers. 

A  phenomenon  somewhat  analogous  to  the  fading  of  the  skin  of  fowls 
during  egg  laying  has  been  observed  in  the  case  of  salmon  during  their 
fresh-water  migration  to  the  spawning  beds  from  the  sea,  during  which 
time  the  animals  starve.  As  described  by  Miss  Newbigin  (1898),  the 
flesh  of  the  fish  has  the  familiar  strong  pink  color  and  the  small  ovaries 
a  yellow-brown  color  when  the  fish  come  from  the  sea.  As  the  re- 
productive organs  develop  the  flesh  becomes  paler  and  the  rapidly  grow- 
ing ovaries  acquire  a  fine  orange-red  color.  The  explanation  of  this 
phenomenon  unquestionably  lies  in  the  mobilization  of  the  fat  stores 
of  the  body  in  the  reproductive  organs  and  the  shed  ova,  rather  than 
in  a  mobilization  of  pigment  itself.  It  is  to  be  remembered  that  the 
fish  are  taking  no  food  whatever  during  their  migration,  and  must 
therefore  draw  upon  every  possible  reserve,  not  only  for  their  own 
needs  but  also  for  the  reproduction  processes  for  which  the  journey  is 
taken.  Essentially  this  view  of  tlie  phenomenon  was  adopted  by  Miss 
Newbigin. 


FUNCTION  OF  CAROTINOIDS  IN  PLANTS,  ANIMALS  ^  275 

For  three-quarters  of  a  century  the  breeders  of  Guernsey  cattle,  one 
of  the  Channel  Island  dairy  breeds,  have  laid  great  emphasis  upon  the 
fact  that  under  comparable  conditions  the  milk  and  butter  from  the^e 
cows  has  a  higher  yellow  color  than  is  produced  by  any  of  the  other 
known  breeds  of  dairy  cattle.  It  is  also  generally  recognized  by  the 
breeders  of  these  cattle  that  a  high  yellow  secretion  by  the  skin  is  re- 
lated to  the  production  of  highly  colored  milk  and  butter.  These  yel- 
low secretions  are  usually  localized  at  certain  parts  of  the  body,  espe- 
cially in  the  ear,  on  the  end  of  the  tail  bone,  and  about  the  udder.  In 
fact,  at  the  present  time  the  official  scale  of  points  for  judging  Guernsey 
cattle  includes  15  points  for  skin  color  on  the  parts  of  the  body  men- 
tioned. In  judging  bulls  a  similar  allowance  is  made  for  high  color  in 
the  ears  and  on  the  tail  and  body  generally  as  indicating  the  ability  of 
the  animal  to  transmit  the  production  of  highly  colored  milk  to  the 
offspring.  Jersey  cattle  show  the  same  characteristics  but  not  to  so 
great  an  extent.  It  should  be  stated,  however,  that  the  ability  of  cows 
of  the  Guernsey  breed  to  transfer  the  carotin  from  their  feed  to  the 
milk  is  not  so  firmly  fixed  in  the  breed  generally  as  the  enthusiastic 
advocates  of  the  breed  would  lead  one  to  believe.  Hill  (1917)  states 
that  on  the  Island  of  Guernsey  itself  there  is  a  marked  difference  be- 
tween the  color  of  the  butter  brought  to  market  from  different  herds. 

There  is  also  a  general  feeling  among  the  Jersey  and  Guernsey  cattle 
breeders  that  abundant  yellow  secretions  localized  on  the  body  indicate 
large  producers  of  butter  fat.  Hooper  (1921),  who  tried  to  correlate 
these  ideas  from  observations  which  he  made  on  about  160  animals, 
could  find  no  relation  between  either  the  amount  or  color  of  the 
secretions  and  the  production  of  either  milk  or  butter  fat,  using  yearly 
production  records  as  the  basis  for  his  conclusions.  The  general  idea 
is  seen  to  be  quite  the  reverse  of  the  relations  found  to  exist  between 
the  color  of  the  skin  of  fowls  and  egg  production.  As  a  matter  of 
fact  if  the  phenomena  of  milk  production,  especially  of  milk  fat  pro- 
duction, and  egg  production  are  related  physiologically  the  correlation 
between  the  production  of  milk  fat  and  the  color  of  the  skin  secretions 
should  be  between  high  production  and  low  skin  color  and  not  between 
high  production  and  highly  pigmented  skin  as  the  breeders  of  Jersey 
and  Guernsey  cattle  seem  to  think.  Furthermore,  by  analogy  with 
the  hen,  the  fresh  cow  or  the  dry  cow  is  not  suited  for  judging  the  fat- 
producing  ability  by  the  amount  or  color  of  the  skin  secretions,  but 
rather  only  the  cow  at  the  close  of  her  lactation  period.  So  far  as 
the  writer  has  been  able  to  ascertain  no  observations  have  ever  been 


276  CAROTINOIDS  AND  RELATED  PIGMENTS 

made  indicating  that  the  yellow  skin  secretions  of  Jersey  and  Guernsey 
cattle  change  their  appearance  with  advance  in  the  lactation  period.  It 
is  perhaps  not  too  hazardous  to  predict  that  it  is  only  along  such  lines 
that  a  correlation  may  be  expected  to  exist  between  skin  pigmentation 
and  butter  fat  production  for  cows  of  the  Channel  Island  breeds.  As 
a  matter  of  fact  Hooper's  data  show  a  slight  indication  of  such  a  cor- 
relation for  one  group  of  cows  but  not  for  the  other  whose  records  and 
skin  colorings  are  recorded.  An  investigation  of  the  theory  from  the 
point  of  view  of  a  fading  of  the  skin  color  during  heavy  production 
might  lead  to  very  profitable  results. 

This  brief  discussion  indicates  the  practical  ends  which  may  be 
served  through  the  occurrence  of  plant  carotinoids  in  the  animal  body. 
The  whole  subject  is  a  fascinating  one  and  offers  as  many  unsolved 
problems  as  any  other  phase  of  experimental  biology  and  biochemistry. 
The  writer  can  not  conclude  this  monograph,  however,  without  inviting 
the  attention  of  the  biochemists  to  this  field  of  work.  The  extension 
of  the  frontiers  of  our  knowledge  regarding  these  pigments  which  are 
so  abundantly  distributed  in  so  many  plants  and  animals  is  certain  to 
prove  a  profitable  as  well  as  an  interesting  undertaking.  Who  can 
predict  the  magnitude  of  importance  of  the  discovery  which  lies  just 
beyond  the  horizon  in  this  or  any  other  expedition  in  the  search  after 
truth? 

Summary 

The  functions  of  the  carotinoids  in  plant  tissues  have  not  been  defi- 
nitely determined.  The  various  theories  which  have  been  advanced 
include  the  following: 

(1)  Carotin  plays  a  role  in  plants  similar  to  that  of  the  hemoglobin 
of  the  blood  ( Arnaud) . 

(2)  Carotin  acts  as  a  reserve  substance  (Zopf,  Kohl). 

(3)  Carotin  shares  in  the  work  of  COg  assimilation  by  acting  in- 
directly as  a  catalyst  for  the  decomposition  of  atmospheric  CO2 
through  its  absorption  of  light  energy  which  it  helps  to  transform  into 
heat  (Kohl). 

(4)  Carotinoids  protect  cell  enzymes  against  the  light  rays  which 
they  absorb  (Went,  Kohl). 

(5)  Carotin  in  flowers  and  fruits  acts  biologically  as  a  lure  for 
insects,  birds  and  other  animals,  in  connection  with  the  spreading  of 
pollen  and  seeds  (Kohl). 


FUNCTION  OF  CAROTINOIDS  IN  PLANTS,  ANIMALS    277 

(6)  Carotinoids  help  regulate  the  oxygen  pressure  in  plant  cells 
through  their  great  affinity  for  this  element  (Willstatter  and  Mieg) . 

(7)  Carotinoids  help  control  the  COg  assimilation  by  controlling  the 
equilibrium  between  chlorophyll  a  and  chlorophyll  b  (Willstatter  and 
Stoll). 

(8)  Carotin  and  xanthophylls  play  a  part  in  photo-synthesis  because 
they  are  believed  to  yield  HCHO  on  photo-oxidation,  xanthophyll  also 
yielding  sugar  (Ewart). 

There  is  no  evidence  to  indicate  that  carotinoids  originate  from 
chlorophyll,  but  it  is  possible  that  both  classes  of  pigment  may  arise 
from  isoprene,  CgHg. 

The  factor  for  lycopin  formation  in  tomatoes  can  be  suppressed  at 
30°  C.  or  above,  the  fruits  forming  only  carotin  and  xanthophylls. 
At  lower  temperatures  all  three  types  of  carotinoids  are  formed.  Syn- 
thesis of  lycopin  is  independent  of  light  but  depends  upon  oxygen,  and 
is  depressed  by  the  conditions  which  accompany  low  catalase  activity 
and  decreased  acidity. 

The  author  believes  that  if  the  carotinoid  pigments  in  animals  pos- 
sess a  definite  function,  this  function  must  be  linked  with  the  physio- 
logical processes  of  the  body,  inasmuch  as  the  carotinoids  are  derived 
from  the  food.  There  are  a  number  of  general  facts,  however,  which 
indicate  that  these  pigments  play  no  definite  role  in  nutrition  or  in 
metabolic  processes,  at  least  in  the  higher  animals. 

A  critical  review  of  the  theories  regarding  the  possible  relation  of 
carotinoids  and  vitamin  A  leads  to  the  conclusion  that  the  substances 
cannot  be  identical.  It  appears  that  there  is  a  fairly  definite  correla- 
tion between  the  occurrence  of  carotinoids  and  vitamin  A  in  plant 
tissues  but  not  in  animal  tissues  or  in  animal  fats.  Animals,  there- 
fore, possess  the  power  to  separate  carotinoids  and  vitamin  A.  Ex- 
periments are  suggested  whereby  this  fact  can  be  further  substantiated. 

Xanthophylls  in  fowls  have  a  definite  function  from  the  standpoint 
of  practical  utility  in  that  there  is  a  correlation  between  low  pigmenta- 
tion of  the  visible  skin  parts  of  certain  breeds  of  fowls  and  high  egg 
production.  The  cause  of  this  phenomenon  is  a  selective  mobilization 
of  pigment  in  the  ova  during  egg  production,  preventing  its  excretion 
by  means  of  the  skin.  An  analogous  phenomenon  occurs  in  the  sal- 
mon during  their  fresh-water  migration  to  the  spawning  beds. 

It  is  generally  believed  by  certain  cattle  breeders  that  abundant 
(carotinoid)  pigmentation  of  the  skin  of  Guernsey  and  Jersey  cattle 


278  CAROTINOIDS  AND  RELATED  PIGMENTS 

is  correlated  with  large  fat  production  in  the  milk.  The  author  sug- 
gests the  possibility  that  if  a  correlation  does  exist  in  this  case  it  is  be- 
tween low  pigmentation  and  high  production  rather  than  the  reverse, 
and  is  analogous  to  that  which  is  found  in  laying  hens. 


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Kohl,  Frederick  G.  1902d.  Ibid., 
page  61. 

Kohl,  Frederick  G.  1902e.  Ibid., 
Chapter  V. 

Kohl,  Frederick  G.  1902f.  Ibid., 
Chapter  VII. 

Kohl,  Frederick  G.  1902g.  Ibid., 
Chapters  VI  and  X. 

Kohl,  Frederick  G.  1902h.  Ibid., 
Chapter  VIII. 

Kohl,  Frederick  G.  1902i.  Ibid., 
Chapter  XL 

Kohl,  Frederick  G.  1902j.  Ibid., 
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Stephenson,  Marjory  1920.  "A  note 
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Stollzner,  W.  1919.  "Ueber  Pseudo 
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INDEX  OF  AUTHOES 


Akutsu,  Dr.,  135,  279. 

Alting,  C,  294. 

Angelucci,  A.,  279. 

Arnaud,  A.,  19,  26,  27,  36,  39,  50,  53, 
58,  78,  83,  88,  179,  180,  199,  200,  202, 
234,  248-251,  263,  264,  276,  279. 

Askenasy,  E.,  31,  94,  95,  107,  279. 

Bachmann,  E.,   114-117,  119,  279. 

Bary  de,  103,  104,  279. 

Bate,  C.  S.,  185,  279. 

Beddard,  F.  E.,  185,  186,  279. 

Behr,   A.,  21,  235,  279. 

Bergh  van  den,  H.  H.  H.,  127-131,  133, 

134,  136,  137,  140,  194,  195,  214,  218, 

225,  270,  279. 
Bertrand,  G.,  71,  114,  115,  279. 
Berzelius,  J.  J.,  31,  34,  49,  56,  58,  279. 
Bidgood,  J.,  67,  69,  71,  72,  74,  75,  279. 
Blakeslee,  A.  F.,  272,  279,  283. 
Blanchard,  R.,  163,  279. 
Blumenthal,  M.,  21,  235,  280. 
BIythe,  A.  W.,  131,  280. 
Boehm,  J.,  49,  280. 
Boeminghaus,  245. 
Bogdanow,  A.,  142,  143,  146,  280. 
Boll,  F.,  280. 
Bonnet,  C,  48,  280. 
Borodin,  J.,  34,  35,  37,  41,  42,  88,  103, 

104,  280. 
Bougarel,  C,  34,  35,  37,  88,  280. 
Bourchadat,   25,   292. 
Boutwell,  P.  W.,  269,  292. 
Broekmeyer,  J.,  127,  129-131,  133,  134, 

136,  137,  140,  194,  195,  218,  225,  270. 
Buell,  M.  v.,  269,  270,  292. 
Burger,  M.,  129,  136,  280. 

Capranica,  S.,  126,  138,  141,  149,  280. 

Caspary,  R.,  103,  104,  280. 

Caventau,  B.,  7,  280. 

Cempert,  I.,  32,  280. 

Charguerand,  A.,  280. 

Chevreul,  M.,  280. 

Cloez,  67,  282. 

Cohn,  F.,  103-105,  109,  280. 

Courchet,  28,  58,  65,  69,  71-75,  78-80,  83, 

86  280 
Coward,  K.  H.,  270,  271,  280. 
Crampton-Simons,  220. 


Cunningham,  J.  T.,  147,  148,  280. 
Czapek,  F.,  18,  49,  94,  95,  97,  280. 

Dastre,  A.,  186,  280. 
Denis,  127,  280. 
Dennert,  E.,  280. 
Desmouliere,  A.,  76,  131,  280. 
Dippel,  L.,  34,  35,  37,  54,  68,  281. 
Dolley,  D.  H.,   134,   135,  245-247,  281. 
Dombrowsky,  Dr.,  189,  281. 
Dorp  van,  W.  A.,  21,  235,  279,  283. 
Drummond,  J.  C,  269-271,  274,  280. 
Duggar,  B.  M.,  76,  79,  82,  83,  85,  86, 
266,  281. 

Eckles,  C.  H.,  28,  88,  128,  129,  132,  135, 
138,  151,  182,  185,  186,  192,  196,  271, 
288,  289. 

Edmond,  H.  D.,  293. 

Ehring,  C,  69,  83,  281. 

Elfving,  F.,  49,  53,  281. 

Englemann,  T.  W.,  263,  281. 

Escher,  H.  H.,  15,  27,  46,  82-85,  88,  90, 
127,  132,  139,  140,  151,  152,  173-176, 
178-182,  190,  193,  197,  201,  224,  233, 
234,  236-239,  245,  281,  294. 

Etti,  C,  22,  281. 

Euler,  H.,  28,  88,  201,  233,  281. 

Ewart,  A.  J.,  48,  52,  53,  265,  277,  281. 

Exner,  F.  and  G.,  281. 

Fenyvessey,  B.,   134,  290. 

Filhol,  E.,  31,  281. 

Findlay,  G.  M.,  133,  281. 

Fintelmann,  H.,  281. 

Fischer,  H.,  182,  196,  281. 

Floresco,  N.,  280. 

Formanek,  J.,  28,  281. 

Frank,  A.  B.,  103,  104,  281. 

Frank,  B.,  51,  55,  282. 

Frank,  C.  A.,  291. 

Frederique,  L.,  161,  282. 

Fremy,  E.,  29-31,  33,  36,  41,  49,  56,  60, 

67,  88,  282,  293. 
Fuchs,  R.  R.,  146,  282. 

Gadow,  H.,  142,  282. 
Gaidukov,  N.,  95,  109,  282, 
Gallerani,  G.,  129,  282. 
Gamble,  F.  W.,  189,  282,  284. 


296 


INDEX  OF  AUTHORS 


297 


Garcin,  A.,  110,  282. 

Gautrelet,  L.,  131,  280. 

Geddes,  P.,  282. 

Gerlach,  M.,  177,  282, 

Gerould,  J.  H.,  186,  196,  282. 

Geyer,  K.,  157,  282. 

Gill,  A.  H.,  77,  79,  81,  87,  220,  282. 

Gobley,  282. 

Goebel,  F.,  7,  282. 

Goerrig,  E.,  53,  57-64,  89,  90,  256,  282. 

Goode,  G.  B.,  183,  282. 

Goppelsroeder,  F.,  70,  282. 

Gorgensen,  I.,  210,  265,  284. 

Graebe,  C,  21,  235,  282. 

Greilach,  P.  H.,  49-52,  282. 

Griffiths,  A.  B.,  120,  159,  160,  282. 

Gross,  E.  G.,  269,  292. 

Guibourt,  55,  282. 

Guignet,  E.,  35,  88,  282. 

Guthrie,  F.  V.,  134,  135,  245,  246,  281. 

Halliburton,  W.  D.,  140,  162,  283. 
Hammarsten,  O.,  129,  283. 
Hannover,   140,  283. 
Hansen,  A.,  32,  49,  50,  68,  74,  78,  95, 

100,  283. 
Harpe  de  la,  C,  21,  235,  283. 
Harris,  J.  A.,  272,  279,  283,  290. 
Hartsen,  F.  A.,  33,  34,  39,  77,  80,  81, 

88    283 
Hashimato,  H.,  135,  283. 
Hasselt  van,  J.  F.  B.,  22,  283. 
Head,  G.  D.,  129,  136,  283. 
Heim,  F.,  161,  283. 
Hering,  T.,  149,  185,  283. 
Herxheimer,  G.,  245,  283. 
Hess,  A.  F.,  130,  135,  139,  193,  194,  283. 
Hildebrand,  F.,  103,  104,  283. 
Hilger,  A.,  69,  283. 
Hill,  C.  L.,  275,  283. 
Hill,  E.  G.,  22,  283. 
Hollstein,  R.,  283. 

Holm,  F.,  14,  23,  125,  126,  150,  177,  283. 
Hooper,  J.  J.,  275,  276,  284. 
Hopkins,  F.  G.,   155,  284. 
Hoyer,  149,  185,  283. 
Hueck,  W.,  133,  249,  284. 
Husemann,  A.,  26,  27,  219,  284. 

Immendorff,  H.,  36,  49,  50,  53,  58,  59, 

69,    81,    88,    284. 
Irving,  A.  A.,  264,  284. 

Jacobsen,  H.  C,  105,  106,  284. 
Johnson,  R.  A.,  129,  136,  283. 
Jolyet,  F.,   161,  284. 

Karsten,  G.,  104,  284. 
Kaup,  W.,  135,  284. 
Keeble,  F.,  282,  284. 


Keegan,  P.  Q,  284. 

Kempster,  H.  L.,  22,  23,  87,  88,  138,  142, 

193,  268,  270,  273,  289. 
Kennedy,   C,    132,    134,   195,   270,   271, 

289. 
Kent,  H.  E.,  269,  292. 
Kidd,  F.,  265,  284. 
Kirchner,  A.,  69,  284. 
Kirkpatrick,  W.  F.,  272,  279,  283. 
Klebs,  G.,  103,  105,  109,  284. 
Klose,  E.,  135,  284. 
Kohl,  F.  G.,  16,  18,  27,  38,  41,  44,  48- 

55,  61,  63,  69,  73,  74,  76,  78-83,  86, 

87,  101-105,   107,   108,   110,   114,   116- 

118,  179,  200,  201,  206,  218,  222,  232, 

250,  263,  264,  276,  284. 
Konrad,  M.,  32,  284. 
Kraemer,  H.,  284. 
Kraus,  C,  33,  34,  60,  285. 
Kraus,   G.,  31-34,  37,  42,  44-47,  49-53, 

60-63,  67,  75,  88,  94,  98,  110,  177,  250, 

285 
Kreibich,    C,   285. 
Kremer,  J.,  160,  285. 
Kressmann,  F.  W.,  57,  285. 
Krukenberg,  C.  F.  W.,  16,  17,  23,  68, 

110,  127,  128,   133;  140,  143-152,  157, 

160-164,  166,  167,  169,  183,  197,  285, 

286. 
Kiihne,  W.,  23,   127,  128,  138,  141-143, 

149,  150,  152,  162,  169,  173,  174,  243, 

286. 
Kutscher,  F.,  110,  286. 
Kylin,  H.,  94-97,  99,  100-102,  286. 

Langen  de,  C.  D.,  294. 

Lendrich,  K.,  286. 

Lewin,  L.,  286. 

Leydig,  F.,  149,  158,  160,  286. 

Lieben,  A.,  14,  125,  150,  177,  289. 

Loisel,  G.,  286. 

Lubarsch,  O.,  133,  134,  286. 

Lubimenko,  V.,  20,  29,  47,  58,  59,  65,  66, 
76,  78,  79,  82,  85-87,  89,  90,  216,  217, 
224,  225,  230,  238,  250,  251,  255,  266, 
286-288. 

MacMunn,  C.  A.,  23,  147,  148,  152,  162- 

164,  166-168,  170,  280,  286. 
McCarrison,  R.,  272,  286. 
Maass,  F.,  135,  286. 
Macaire-Prinsep,  55,  60,  286. 
Macchiati,  L.,  158,  286. 
Magnan,  A.,  149,  287. 
Maly,  R.,  23,  162,  163,  287. 
Marchlewski,  L.,  236,  287. 
Marquart,  L.,  66,  287. 
Matzdorff,  C,  185,  287. 
Medola,  R.,  155,  287.  '      '  ' 

Mer,  E.,  56,  287. 


298 


INDEX  OF  AUTHORS 


Merejowski  de,  C,  146,  147,  162,  164, 

166,  167,  169,  287. 
Meschede,  F.,  134,  287. 
Meyer,  A.  B,  287. 
Mieg,  W.,  27,  34,  36,  38,  39,  45-47,  84, 

88,  89,   129,   139,    174,   175,   179,  202, 

203,  209-211,  225,  233,  234,  236,  237, 

264,  277,  294. 
Miethe,  A.,  286. 
Millardet,  A.,  82,  83,  90,  94-96.  104,  111, 

122,  285,  287. 
Miura,  K.,  135,  287. 
Mobius,  K.,  69,  185,  287. 
Molisch,  H.,  51,  52,  54,  55,  58,  59,  65, 

72,  74,  78-82,  85,  94-97,  101-107,  111, 
115,  240,  241,  243,  287. 

Monier-Williams,  G.  W.,  88,  287. 

Montanari,  C,  83,  287. 

Monteverde,  N.  A.,  41,  42,  47,  49,  50, 

65,  66,  76,  78-80,  82,  85,  87,  90,  98, 

111,  112,  216,  217,  222,  225,  230,  238, 

250,  251,  255,  287,  288. 
Moro,  E.,  135,  189,  288. 
Moseley,  H.  N.,  161,  288. 
Muller,  P.,  114,  127,  129-131,  133,  134, 

136,  137,  140,  194,  195,  214,  218,  225, 

270   279. 
Muller,  J.^  114,  115,  288. 
Muller,  N.  J.  C,  288. 
Myers,  V.  C,  130,  135,  136,  193,  194,  283. 

Nageli,  107,  111,  288. 

Nebelung,  H.,   100,  101,  103,  104,  107, 

109,  111,  288. 
Negri  de,  A.  and  G.,  78,  83,  90,  288. 
Nelson,  E.  M.,  270,  292. 
Neumann,  E.,  23,  244,  288. 
Newbigin,  M.  I.,  148,  162-166,  168,  184, 

185,  192,  195-197.  263,  275,  288. 
Noorden  von,  C,  136,  288. 
Nordenson,  E.,  28,  88,  201,  233,  281. 

Overbeck,  A.,  121,  288. 

Pabst,  T.,  69,  82,  288. 

Page,  H.  J.,  94-99,  103,  104,  122,  215, 

226,  230,  257,  258,  294. 
Palmer,  L.  S.,  15,  22,  23,  26,  28,  41,  72, 

73,  87,  88,  127-132,  134,  135,  138,  139, 
142,  151,  152,  182,  185,  186,  192,  193, 
195-198,  207,  208,  219,  268,  270,  271, 
273,  288,  289. 

Palozzi,  A.,  176,  291. 
Passerini,  N.,  83,  289. 
Phipson,  T.  L.,  48,  49,  289. 
Physalix,  C,  158,  177,  289. 
Piccolo,  G.,  14,  125,  150,  177,  289. 
Podiapolsky,  P.,  160,  289. 
Poirault,  G.,  71,  114,  115,  279. 
Pouchet,  G.,  161,  164,  289. 


Poulton,  E.  B.,  155-158,  171,  183,  184, 

185,  188,  189,  197,  289. 
Pringsheim,  49,  52,  59,  289. 
Przibram,  H.,  186,  289,  290. 
Pummerer,  R.,  21,  235,  290. 

Rafarin,  290. 

Raj  us,  J.,  48,  290. 

Regnard,  P.,  161,  284. 

Reinhart,  A.,  129,  136,  280. 

Reinke,  J.,  42,  94-96,  100,  101,  111,  290. 

Riddle,  0.,  290. 

Rosanoff,  S.,  94,  95,  122,  290. 

Rose,  H.,  182,  196,  281. 

Rosenheim,  0.,  270,  274,  290. 

Rosin,  H.,  134,  290. 

Rostafinski,  J.,   103-105,  290. 

Rywosh,  S.,  290. 

Sachs,  J.,  49,  56,  290. 

Sachsse,  R.,  35,  290. 

Salomon,  H.,  129,  135,  136,  290. 

Samson,  M.,  127,  290. 

Sauermann,   Dr.,   187-189,   197,  290. 

Schimper,  A.  W.  F.,  28,  65,  68,  75,  79- 

81,  83,  290. 
Schmidt,  A.,  127,  290. 
Schmidt,  F.,  290. 
Schneider,  G.,  186,  290. 
Schneider,  J.  G.,  185,  290. 
Schroeter,  J.,  119,  120,  291. 
Schrotter  von,  H.,  120,  291. 
Schrotter-Kristelli,   H.  R.,   17,   72,   78, 

86,  291. 
Schubler,  G.,  291. 
Schuler,  0.,  291. 
Schulze,  P.,  159,  291. 
Schunck,  C.  A.,  31,  39-44,  46,  52,  53,  61, 

69,  70,  72,  73,  81,  83,  139,  140,  173, 

180,  291. 
Schiissler,  135,  291. 
Schut,  H.,  294. 
Schutt,  F.,  108,  291. 
Schwalbe,  G.,  141,  291. 
Sehrt,  E.,  133,  243,  291. 
Sell,  M.  T.,  269,  270,  292. 
Serono,  C.,  139,  176,  291. 
Sewell,  P.,  291. 
Shenkling-Prevot,  291. 
Sherndal,  A.  E.,  236,  291. 
Sikar,  A.  P.,  22,  283. 
Smith,  F.,  135,  291. 
Smith,  J.  L.,  245,  291. 
Snapper,  J.,  128,  129,  136,  194. 
Sorby,  H.  C,  31,  33,  34,  37,  39,  41,  44, 

49,    60,   63,    68,    88,   95-97,    100,    101, 

111-113,  116,  158,  159,  291. 
Staats,  G.,  58,  60,  292. 
Stiideler,  G.,  138,  292. 
Stahl,  E.,  263,  292. 


INDEX  OF  AUTHORS 


299 


Steenbock,  H,  206,  269-271,  292. 
Stenger,  E.,  286. 
Stephenson,  M.,  219,  270,  292. 
Stiles,  W.,  210,  284. 
Stokes,   G.   G.,   29-31,  33,  37,   88,  292. 
StoU,  A.,  39,  45-47,  53,  64,  221,  225,  228, 
231,  251-256,  260,  264,  265,  277,  294. 
Stollzner,-  W.,   135,  292. 

Tammes,  T.,  48,  51,  52,  54,  57-59,  69, 
72-74,  78-81,  83,  86,  96,  101-105,  107, 
111,  240,  292. 

Thiele,  J.,  21,  292. 

Thrun,  W.  E.,  26,  219,  289. 

Thudichum,  J.  L.  W.,  14-16,  22,  54, 
75,  77,  79-82,  87,  126-129,  138,  292. 

Timiriazeff,  C,  31,  41,  49,  292. 

Tobler,  G.  and  F.,  72,  79,  86,  266,  292. 

Treub,  M.,  32,  293. 

Tschirch,  A.,  37-39,  41,  49,  51,  54,  59, 
61,  69,  70,  73,  74,  76,  78,  80-82, 
293. 

Tswett,  M.,  7,  15,  19,  20,  24,  28,  30,  35, 
37,  38,  41-45,  51,  55-58,  60-66,  70,  71, 
80,  88-90,  94-100,  102,  106,  145,  177, 
190,  203,  204,  209,  216,  218,  219,  224, 
226,  227,  229,  293. 

Ude,  H.,  293. 
Umber,  129,  136,  293. 
Urech,  F.,  155,  293. 

Valenciennes,  A.,  293. 

Vauquelin,  25,  293. 

Veitch,  H.  J.,  293. 

Verne,  J.,  163,  171,  179-181,  293. 


Villard,  J.,   186,  293. 
Virchow,  R.,  23,  293. 

Wachenroder,  H.,  25,  88,  293. 

Waelchi,   G.,  142,  293. 

Wagner,  H.,  133,  147,  148,  286. 

Warner,  D.  E.,  272,  279,  283,  293. 

Wells,  H.  G.,  244,  294. 

Went,  264,  276,  294. 

Westwood,  I.  0.,  185,  279. 

Wheldale,   M.,   21,   57,  294. 

Wiehuizen,  F.  E.,  272,  294. 

Wiesner.  J.,  32,  49,  52,  53,  294. 

Wille,  N.,  109,  294. 

Willstatter,  R.,  15,  27,  28,  36-39,  43,  45- 
47,  53,  64,  82-85,  88-90,  94-100,  103, 
104,  122,  129,  139,  140,  152,  173-180, 
182,  190,  193,  197,  202,  203,  209,  212, 
213,  215,  218,  221,  224-226,  228,  230- 
234,  236-239,  251-260,  254-266,  277, 294. 

Windaus,  A.,  206,  294. 

Wirth,  F.,  69,  294. 

Wisselingh  van,  C,  28,  51,  55,  69,  71- 
74,  77,  78,  80-82,  85,  86,  95,  96,  101- 
106,  110,  111,  114-119,  237,  240,  241, 
294. 

Wittich,  von,  110,  149,  185,  294. 

Wittmack,  L.,  28,  294. 

Wurm,  Dr.,  142,  294. 

Zeise,  25-27,  36,  88,  294. 

Zilva,  S.  S.,  269,  281. 

Zoia,  L.,  129,  294. 

Zopf,  W.,  19,  22,  23,  83,  104-107,  109- 

121,   159,  160,   162-164,  177,  183,  197, 

263,  276,  294,  295. 


INDEX  OF  SUBJECTS 


a'  and  a' '  xanthophylls  of  Tswett,  44. 

Abies,  65;  Abies  excelsa,  52. 

Abutilon  Darwinii  Hook,  72;  Abutilon 
megopotamicum,  73;  Abutilon  ner- 
vosum, 54,  73. 

Acacia  leaves,  carotin  content  of,  249. 

Acenaphtylene,  a  non-carotinoid  hydro- 
carbon pigment,  21,  235. 

Acerata,  161. 

Acer  campestris,  58;  Acer  platanoides, 
58,  249;  Acer  pseudoplaianus,  36,  58, 
249. 

Achinophlocus  angustijolius  Becc,  A. 
macarthurii  Becc,  77. 

Achnanthidium  lanceolatum,  107. 

Acidity,  relation  of  to  lycopin  forma- 
tion in  tomatoes,  267. 

Acid  microchemical  crystallization 
method,   51. 

Adipose  tissue,  color  of  in  laying  caro- 
tinoid-free  hens  after  xanthophyll 
feeding,  273;  pigments  of,  14,  132, 
133,  151. 

Adipose  tissue  pigments,  experiments 
on  origin  of,  190. 

Adipose  tissue,  quantitative  estimation 
of  carotinoids  in,  258. 

Adonis  vemalis,  73. 

Adrenals,  carotinoids  of,  133. 

Adsorption  properties  of  carotinoids, 
43,  219,  226. 

Aecidia  spores,  114,  116. 

Aesculus  hippo castanum,  58,  62,  117, 
249,  255. 

Aethalium  septicum,  119. 

Afzelia  Cuazensis,  86. 

Agleonema  commutatum  Shott.,  78. 

Agleonema  fruits,  carotinoids  in,  77. 

Ailanthus  gladulosa,  251. 

Albizzia  Julibrissin,  251. 

Alder,  black,  autumn  pigments  of,  58. 

Alfalfa  leaves,  pigments  of,  45. 

Alfonsia  oleifera  Humb.,  77. 

Alizarin,  use  of  for  quantitative  esti- 
mation of  carotinoids,  253. 

Alkalis,  effect  of  on  chlorophylls,  32; 
on  carotinoids,  32,  219,  225,  231. 

Alkali  microchemical  crystallization 
method,  51,  52,  54,  241;  non- 
specificity  of  for  carotin,  59. 


Alligator,  yellow  pigment  in  skin  of, 
150. 

Allium  siculum,  74. 

Alnv^  glutinosa,  58. 

Aloes,  58;  Aloe  verrucosa,  red  caro- 
tinoid  in  winter  leaves  of,  65,  72. 

Alyssum  saxatile,  73, 

Amorphous  carotin,  effect  of  admixed 
lipoids  on  solubility  of,  218. 

Amorphous  xanthophyll,  effect  of  ad- 
mixed lipoids  on  solubility  of,  225. 

Ampelopsis  hederaceae,  78. 

Amphibians,  carotinoids  in,  148,  149, 
153. 

Anabaena  flos  aquae  Bub.,  111. 

Anemalocera  Patersoni,  164. 

Animal  fats,  color  and  vitamin  content 
of,  270. 

Animal  tissues,  color  of  carotinoid 
granules  in  after  staining,  245-247; 
effect  of  oxidation  on,  246;  identifi- 
cation of  carotinoids  in,  242-247; 
state  of  carotinoids  in,  242. 

Annatto  seeds,  pigment  of,  15,  21 ;  ef- 
fect  of  feeding  to  fowls,  22,  138. 

Aniedon  rosacea,  167. 

Anthocyanins  in  autumn  leaf  colora- 
tions, 57;  in  flowers,  66,  67,  68. 

Anthoxanthins  in  flowers,  66. 

Apanteles  flaviconchae,  186. 

Aphidoluteine,  158. 

Aphids,  anthocyanin-like  pigment  in, 
158;  carotinoids  in,  158,  171. 

Aplysina  aerophoba,  170. 

Apricot,  carotinoids  in,  76. 

Aprosmictu^  melanurus,  143,  145. 

Aralia,  58. 

Araroth,  red  pigment  in  feathers,  143. 

Arils,  carotinoids  in,  86-88,  90. 

Arbor  Vitae,  autumn  pigments  of,  59, 
66,  216 ;  carotin  and  xanthophyll  con- 
tent of,  251. 

Archonthophoenix  Alexandrae  H 
Wendl.,  77. 

Arcyria  punicea  Pers.,  119;  A.  nutans 
BiiU. 

Areca  Alicae  W.  Hill,  77. 

Arenicola  piscatorium,  168. 

Armeria  vulgaris,  74. 

Arnoglossus  megastoma,  147. 


300 


INDEX  OF  SUBJECTS 


301 


Artherina  presbyter,  147. 

Arum  italicum,  79;  Arum  orientale,  77. 

Arundinana  japonica,  251. 

Asclepias  Curassavica  L.,  72. 

Ascobolus  species,  116. 

Ascomycetes,  116-118. 

Ascophyllum  nodosum,  95. 

Ash,  autumn  pigments  of,  58,  60. 

Ash,  mountain,  pigment  of  red  autumn 
leaves  of,  56;  carotinoids  in  fruits 
of,  81. 

Asparagus  berries,  carotin  in,  77. 

Aspergillus  giganteus,  119. 

Asphodel,  carotin  in  flowers  of,  72. 

Asphodelus  cerasifer  L.,  72. 

Astacus  fluviatilis,  162;  Astacus  no- 
hilis,  164. 

Aster  species,  73. 

Aster acanthion  glacialis,  166. 

Asterina  gibbosa,  166. 

Asteroids,  165,  166. 

Astrogriseine,   167. 

Astrospecten  aurantiacus,  166. 

Astroviolettine,   167. 

Astroviridine,  165,  167. 

Atropa  belladonna,  74. 

Aucuba  japonica  Thunb.,  54. 

"Ausschuttelungs"  method  of  Kraus,  31. 

Autumn  leaf  pigments,  31,  33,  55-66; 
as  due  to  chlorophyll  migration,  56; 
as  due  to  non-carotinoids,  57;  plu- 
rality of  carotinoids  in,  59-64,  89. 

Autumn  leaves,  difficulties  of  deter- 
mination of  carotinoids  in,  256. 

Autumn  reddening  of  leaves,  65,  66, 
90. 

Autumn-xanthin,  60. 

Autumn  xanthophylls  of  Tswett,  prop- 
erties of,  62;  relation  of  to  carotin, 
63. 

Avena  sativa,  88. 

a'  xanthophyll  of  Kohl,  41 ;  in  autumn 
leaves,  61 ;  of  Tschirch,  59 ;  of  Tswett, 
44,  45. 

Azulene,  a  blue  hydrocarbon,  236. 

Bacteria,  carotinoids  in,  119-122,   123. 

Bacteria,  study  of  carotinoids  in  as 
means  of  establishing  function,  93, 
119,  124. 

Bacterium  Chrysogloia,  120,  121;  B. 
egregium,  120,  123;  B.  prodigiosus, 
120;  B.  xanthinum,  120. 

Bacillariea,  106-108. 

Balsam  apple,  carotin  in  flowers  of, 
72;  lycopin  in  fruit  of,  79;  in  arils, 
86;  Balsam  pear,  lycopin  and  caro- 
tin in,  76. 

Balsam,  pigment  of  etiolated  leaves,  52. 

Balsamina  hortensis,  D.  C,  52. 


Barberry  leaves,  red  autumn  pigment 
of,  56;  fruits,  carotinoids  in,  76. 

Barley  leaves,  etiolated,  pigments  of, 
52;  barley  grain,  carotinoids  in,  88. 

Bangia  species,  101. 

Bangiales,  101. 

Barbu-s  fluviatilis,  147. 

Basidiomycetes,  113-116,  123;  chryso- 
phanic  acid  in,  22. 

Batrachospermum  monilijorme,  100, 
101. 

Batracians,  149. 

Bean  leaves,  carotin  content  of,  249. 

Beech,  carotin  in  yellow  lautumn  leaves 
of,  59;  autumn-xanthin  in,  60. 

Beet,  red  {Beta  vulgaris),  red  pigment 
of  changing  to  yellow,  28;  quantity 
of  carotin  in  leaves  of,  249. 

Beetles,  carotinoids  in  tegument  of, 
158-160,  171. 

Belladonna,  pigments  in  flowers  of,  74. 

Bellwort,  carotinoids  in  flowers  of,  74. 

Belone  rostrata,  147. 

Berberis  vulgaris,  56,  76. 

Betula  alba,  58. 

Betula  species  (Birch),  54. 

Benzene,  part  played  by  in  Kraus 
separation,  31,  32. 

Bilirubin,  23;  in  blood  serum,  128,  129. 

Birch,  carotinoids  in  naturally  yellow 
leaves  of,  54;  in  autumn  leaves  of, 
58. 

Birds,  carotinoids  in,  137-145,  152;  prob- 
lems in  connection  with,  137,  138; 
retinal  pigments  of,  140-142,  152. 

Birds  of  paradise,  pigments  in  feathers 
of,  143-145. 

Bird-of-Paradise  flower,  carotinoids  in, 
74. 

Bittersweet,  climbing,  autumn  pigments 
of,  58;  pigments  in  fruit  of,  81. 

Bixa  orellana,  21. 

Bixin,  pigment  of  annatto  seeds,  21; 
composition  and  properties  of,  22; 
effect  of  feeding  to  fowls,  22. 

Black  Bryony,  carotinoids  in  fruit  of, 
80. 

Bladder  Senna,  carotinoids  in,  73. 

Blazing  Star,  carotinoids  in,  74. 

Blood  algae,  carotinoids  in,  92,  103- 
106;  factors  governing  pigment  for- 
mation in,  105. 

Blood  exudates,  haematoidin  in,  23. 

Blood  serum  carotinoids,  effect  of  diet 
on,  129,  130,  190,  191,  197;  method 
of  distinguishing  from  bilirubin,  128; 
manner  of  carrying,  207,  208;  meth- 
ods of  extraction,  207. 

Blood  serum  of  cattle,  xanthophyll  in, 
129;    of  fowls,  xanthophyll   in,   140, 


302 


INDEX  OF  SUBJECTS 


152;  of  humans,  carotin  and  xantho- 
phyll  in,  130,  151;  of  horse,  carotin 
and  bilirubin  in,  129,  151;  of  new 
born  calf,  absence  of  carotinoids 
from,  129,  151;  of  various  mammals, 
pigments  in,  131,  151. 

Blood  serum,  pigment  of,  14,  15,  127- 
131,  151;  quantitative  estimation  of 
carotinoids   in,  259,   260. 

Blue-green  algae,  carotinoids  in,  110- 
112,   123. 

Blue  hydrocarbon,  236. 

Blue  pigment  of  Crustacea,  transfor- 
mation of  into  lipochrome,  a  col- 
loidal phenomenon,   165. 

Bombinator  igneus,   150. 

Bombyx  mori,  157. 

Botrydium,  104. 

Box  tree  leaves,  carotin  content  of,  249. 

Bracket  fungi,   carotinoids  in,  113-114. 

Brassica  campestris  L.,  29,  87;  Brassica 
nigra,  87;  Brassica  oleijera,  249; 
Brassica  Rapa  L.,  29. 

Brittle-stars,  carotinoids  in,  165,  167. 

Broom  flower,  xanthophylls  in,  72,  73. 

Broussonetia  papyrijea,  58. 

Brown  sea-weeds,  carotinoids  in,  93- 
100,  122;  isolation  of  carotinoids 
from,  215. 

Bryonia  dioica  (Bryony),  carotinoids 
in  fruit  of,  75. 

Buckeye,  autumn  pigments  of,  58,  62. 

Bujo  calamita,  149;  B.  viridis,  149,  185; 
B.  vulgaris,  149. 

Bugs,  carotin  in,  158,  171,  177. 

Bugula  neritina,  168. 

Bulbine  semibarbata,  74. 

Bulbochaete,  104;  Bulbochaete  seti- 
gera,  104. 

Bulgaria  inquinans  (polymorpha) ,  117. 

Bullfinch  feathers,  pigments  of,  143,  144. 

Buphthalmum  salicifolium,  73. 

Butter,  pigment  of,  14,  131,  132,  151; 
seasonal  variation  of  anti-edema  sub- 
stance in,  272. 

Buttercups,  crystalline  carotin  from, 
69;  cause  of  oily  appearance  of,  69; 
xanthophylls  in,  73,  74. 

Butter  fat,  carotin  and  vitamin  con- 
tent of,  269,  270;  effect  of  decoloriz- 
ing on  vitamin  content  of,  270;  ex- 
periments on  origin  of  color  of,  190- 
192,  197;  quantitative  estimation  of 
carotinoids  in,  258,  260. 

Butter  fat  carotin,  adsorption  of  by 
charcoal,  219. 

Butterflies,  wing  colors  of,  154,  155. 

Butterfly  larvae,  carotinoids  in,  154-158, 
171 ;  pupae,  carotinoids  in,  154-158, 
171. 


Buxus  sempervirens  (box  tree),  red 
winter  pigment  of,  65,  249. 

B.  xanthophyll,  40-42;  blue  color  re- 
action of  with  acids,  40,  42;  acid 
derivative  of  in  autumn  leaves,  61. 

/3  xanthophyll  of  Kohl,  44;  in  autumn 
leaves,  61;  in  diatoms,  107;  of 
Tschirch,  41,  54,  59;  of  Tswett,  44, 
70. 

Cacalia  coccinea,  73. 

Cacatura  roseicapilla,  144. 

Calabazilla,  carotinoids  in  flowers  of, 
73. 

Calceolaria  rugosa  Hook.,  72. 

Calendula  arvensis  L.,  72;  Calendula 
officinale  L.,  69,  72_. 

Calf  blood,  absence  o'f  carotinoids  from 
new  born,  129,  267. 

California  poppy,  68,  74. 

Callithamnion  hiemale,  101. 

Callopeliis   quadrilineatis   Pallas,   150. 

Calocera  viscosa,  114,  115;  C.  cornea, 
114,  115;  Cpalmata,  114,  115. 

Calothrix  species,  HI. 

Caltha  palustris,  73. 

Calurus  auriceps,  144. 

Calyptrocalix  spicatus   Blume,   77. 

Camaelon  vulgaris,  150. 

Camelon  skin,  carotinoids  in,  150,  153. 

Camouflage,  carotinoids  and  detection 
of,  7. 

Campethera  nubica,  145. 

Canary  bird,  xanthophyll  in  yellow 
feathers  of,  145;  effect  of  feeding 
cayenne  pepper  to,  187,  197. 

Cancer  pagurus,  164. 

Cannabis  sativa,  52,  87,  249. 

Capillary  method  of  pigment  analysis, 
70. 

Capsicum  annum,  69,  82,  188. 

Carabus  auratus,  159. 

Cardinalis  virginianus,  144. 

Cardinal,  pigments  in  feathers  of,  143, 
144. 

Carotene,  as  spelling  for  carotin,  19. 

Carotin-albumin  complex  in  blood,  15, 
128;  method  of  isolation  of,  208,  209; 
significance  of  in  determining  breed 
differences  in  pigmentation  of  cattle, 
192;  significance  of  in  formation  of 
milk  fat,  209. 

Carotin,  adsorption  by  mercury  salts, 
15;  alleged  oxidation  of  to  xantho- 
phyll, 264;  chromatophor  group  in, 
235;  extraction  of  from  plants,  251, 
252;  general  properties  of,  25-28; 
hydrocarbon  nature  of,  26-28;  iodine 
derivatives  of,  27,  233,  234;  isola- 
tion  of   from   animal   fat,   204-206; 


INDEX  OF  SUBJECTS 


303 


from  blood  serum,  206-209;  from 
carrots,  25,  26,  200-202;  from  green 
leaves,  202-204;  microchemical  iden- 
tification, of  in  plant  tissues,  241 ; 
odor  of,  26,  232,  233;  possible  rela- 
tion of  to  cymenes,  235;  pronuncia- 
tion of,  9;  relation  of  to  xantho- 
phylls,  24;  relationship  of  to  vita- 
min A,  269-271 ;  separation  of  from 
xanthophylls,  252,  253;  specific  ro- 
tation of,  233;  structure  of,  234, 
235. 

Carotin  content  of  various  plants,  249, 

251,  255. 
Carotin  crystals,  color  and  form  of, 
232;  color  reactions  of,  233;  effect  of 
oxidation  on  solubility  of,  218;  halo- 
gen derivatives  of,  233,  234;  oxygen 
absorption  of,  233;  solubility  of,  232, 
233. 

Carotin  function  in  plants,  as  aid  in 
photo-synthesis,  265,  277;  as  enzyme 
protectors,  264,  276;  as  lure  for  in- 
sects, etc.,  264,  276;  as  regulators  of 

•  oxygen  pressure,  264,  277;  as  reserve 
substances,  263,  276;  as  respiratory 
pigments,  263,  276;  in  CO2  assimila- 
tion, 265,  277. 

Carotin  solutions,  effect  of  acids  on 
color  of,  219;  relative  color  of  com- 
pared to  xanthophyll,  225;  proper- 
ties of,  218-222. 

Carotinemia,  194. 

Carotinins,  basic  compounds  of,  19,  24, 
117,  118;  in  bacteria,  121;  in  beetles, 
171;  in  blood  algae,  105,  106,  123;  in 
cup  fungi,  117;  in  Euglena,  110,  123. 

Carotinoid,  origin  of  name,  19,  88;  pro- 
nunciation of,  9;  suitability  of  name 
for   animal  lipochromes,  20. 

Carotinoid-free  fowls,  experiments  with, 
193,  268;  rapidity  of  coloration  of 
with  xanthophyll-rich  feeds,  193. 

Carotinoid-free  egg  yolk,  vitamin  con- 
tent of,  270. 

Carotinoid-free  mammals,  195;  possible 
cause  of,  196. 

Carotinoids,  decline  of  in  leaves  in  au- 
tumn, 61,  64;  fate  of  in  digestion, 
196;  in  flowers,  66-75;  in  fruits,  75- 
85,  90;  origin  of  in  plants,  266; 
quantitative  estimation  of,  248-261. 

Carotinoids  of  animals,  chemical  rela- 
tions of  to  carotinoids  of  plants,  173- 
181;  biological  relations  of  to  caro- 
tinoids of  plants,  182-198. 

Carotinosis,   135. 

Carpellary  tissue  of  seeds,  pigments  of, 
90. 

Carpinus  Betulus,  58, 


Carrots,  carotin  content  of  during 
starch  formation,  266;  effect  of  feed- 
ing to  cows  on  color  of  butter,  191 ; 
effect  of  feeding  to  hens  on  color 
of  egg  yolk,  193,  271. 

Carrot  root,  pigment  of  as  the  first 
crystalline  carotinoid,  18,  25;  xan- 
thophyll in,  28,  45,  88. 

Cassidae,  158. 

Cat  blood,  effect  of  injecting  xantho- 
phyll on  pigments  of,  131. 

Catalase,  relation  of  to  lycopin  for- 
mation in  tomatoes,  267. 

Caterpillar,  lack  of  lipochrome  in  blue- 
mutant,  186. 

Caterpillars,  carotinoids  in,  156-158; 
color  in  tegument  of  influenced  by 
food,  188. 

Caterpillar  eggs,  pigment  of  derived 
from  haemolymph,  158. 

Caterpillar  haemolymph,  association  of 
carotinoid  in  with  protein,  156;  sex- 
ual difference  in  pigmentation  of, 
157. 

Catinga  coendea,  144. 

Cattle,  color  of  skin  secretion  in  re- 
lation to  fat  production,  275;  skin 
color  of  as  affected  by  breed,  275. 

Cave  animals,  color  of  in  relation  to 
cave  plants,  186. 

Cedar,  red,  autumn  pigments  of,  59. 

Celandine  poppy,  carotin  and  xantho- 
phylls in,  72. 

Celastrus  scandens,  58. 

C.  Elpenor,  157. 

Cephaleunis  albidus,  104;  C.  minimus, 
104;  C.  (Mycoidea)  leavis,  104;  C. 
parasiticus,  104;   C.  solutus,  104. 

Cephalothecium,  119. 

Ceramium  rubrum,,  101 ;  Ceram.ium  dia- 
phanum,  101. 

Certhiola  mexicana,  145. 

Certium  tropis,  108;  C.  jusv^,  108;  C. 
fur  CO,  108. 

Chantransia  species,  101. 

Chara  jragilis,  102. 

Charales,  102. 

Charlock,   white,  xanthophylls  in,  73. 

Cheiranthu^  cheira  L.,  72. 

C helidonium,  maju^  L.,  72. 

Cherries,  erthrophyll  of,  34;  Pitanga  or 
Surinam  cherry,  anthocyanin  and 
carotinoids  in,  76. 

Cherry,  Sweet  Mazzard,  autumn  pig- 
ments of,  58.. 

Cherry  tree  leaves,  red  autumn  pig- 
ments of,  56. 

Chestnut,  autumn  pigments  of,  58. 

Chestnut  leaves,  carotin  content  of, 
249. 


304 


INDEX  OF  SUBJECTS 


Chinese  Lantern  Plant,  carotinoids  in 
flowers  of,  74;  in  fruits,  80. 

Chlorella  protothecoides,  104;  C.  barie- 
gata,  104. 

Chlorine  derivative  of  carotin,  26. 

Chlorogon  in  etiolated  leaves,  49. 

Chloronerpes  aurulentus,  145;  C.  Kir- 
kii,  145. 

Chlorophane  in  bird  retinas,  141 ;  prop- 
erties of,  141. 

Chlorophyceae,  101,  102-106,  table 
showing  species  containing  carotin- 
oids, 104. 

Chlorophyll,  separation  of  from  yellow 
pigments,  30,  31,  226-228,  252. 

Chlorophyllins  in  brown  algae,  97. 

Chloroplastids,  pigments  of,  29-48; 
plurality  of  carotinoids  in,  30-48,  41, 
88,  89. 

Cholechrome  in  mollusc  livers,  rela- 
tion of  to  bilirubin  and  lipochrome, 
167. 

Cholesterol,  removal  of  from  unsa- 
ponifiable  matter,  206. 

Chondrosia  renijormis,  170. 

Chondrus  crispus,  100,  101. 

Chorda  filum,  96. 

Chromatogram,  description  of,  43,  226; 
pronunciation  of,  9. 

Chromatographic  analysis  of  chloro- 
phylls and  xanthophylls,  226-228. 

Chromatophores,  146,  150. 

Chromogens  of  leaf,  yellow  color  of 
with  alkali,  44. 

Chromoleucites,  68. 

Chromolipoid,  origin  of  name,  18;  pro- 
nunciation of,  9. 

Chromulina  (Chromophyton)  Rosa- 
no  fii,  109. 

Chroococcaceae,  111. 

Chrysanthemum,  72 ;  Chrysanthemum 
frutescens,  73. 

Chrysochlorophyll,  109. 

Chrysochrome,  109. 

Chrysomela  polita,  159;  C.  varians, 
159. 

Chrysomelidae,  18,  159. 

Chrysomonidina  Stein.,  109. 

Chrysophanic  acid,  21;  in  fungi,  22. 

Chrysophyll,  33,  36,  40,  42,  173;  rela- 
tion of  to  carotin  and  xanthophyll, 
34. 

Chrysoptilus  punctigula,  145. 

Chrysoquinone,  104. 

Chrysotannis  in  autumn  leaves,  60. 

Chrysoxanthophyll,  109. 

Chy  Iridium,  118. 

Cinnamylidenindene,  a  non-carotinoid 
hydrocarbon  pigment,  21,  235. 

Cirassius  auratus,  147,  152. 


Cirratulus  tentaculatus,  168;  C.  cit' 
ratus,  168. 

Cissu^  quinquefolia,  249. 

Citrus  aurantium,  81. 

Citrus  limonum,  78. 

Cladophora  glomerata,  104. 

Clavaria  jusiformis,  113. 

Clematis  vitalba,  251. 

Clivia  miniata,  Regel,  73. 

Clivias,  76. 

Club  Moss,  autumn  pigments  of,  59. 

Clupea  narengus,  147. 

Clythra  quadripunctata,  159. 

Coccinella  quinquepunctata,  159;  C. 
septempunctata,  159. 

Coccinellidae,  18,  159. 

Cocoons,  relation  of  color  of  to  caro- 
tinoids in  insects,  186. 

Cocospongia,  170. 

Codliver  oil,  color  and  vitamin  con- 
tent of,  269. 

Coelenterates,  lack  of  carotinoids 
among  brilliant  colors  of,  169. 

Colaptes  auratus,  145;  C.  olivaceus,  145. 

Coleoptera,  155,  158-160. 

Coleopterin,  159,  160. 

Coleosporium  Pulsatilla  Strauss,  114. 

Coleus,  pigment  of,  15,  54. 

Colias  (Eurymus)  Philadice,  186. 

Colloidal  carotin,  218;  colloidal  xan- 
thophyll, 225. 

Colloidal  phenomena  involved  in  blood 
serum   carotinoids,   131. 

Colloidal  state  of  carotinoids  in  cer- 
tain solutions,  43,  89. 

Color,  structural  vs.  pigmented,  7. 

Colostrum  milk,  pigments  of  fat  of  in 
various  animals,   132. 

Coltsfoot,  xanthophylls  in,  73. 

Colutea  media,  73. 

Cone  Flower,  carotinoids  in,  74. 

Conifers,  58. 

Conjugatae,  104. 

Convalaria  majalis,  58. 

Copepods,  blue  colors  in,  164. 

Corallina  officinalis,  101. 

Coriosulfurine,  as  cause  of  yellow 
feathers,  144,  145;  relation  of  to  xan- 
thophyll P,  145. 

Corn,  pigmentation  and  vitamin  con- 
tent of,  269. 

Corn,  yellow,  carotinoids  of,  87,  91; 
effect  on  color  of  butter  of  feeding 
to  cows,  191. 

Corpus  luteum,  pigment  of,  14,  23, 
125-127,  150,  151,  177-179,  180;  prop- 
erties and  isolation  of,  178,  179. 

Corydalis  lutea  D.  C,  73. 

Corylus  Avellana,  58. 

Cotoin,  21. 


INDEX  OF  SUBJECTS 


305 


C otoneasters,  76. 

Cottonseed  meal,  pigment  of,  21. 

Cottonseed  oil,  pigments  of,  87. 

Cowslip,  carotin  in,  72;  xanthophylls 
in,  73. 

Crab,  pigment  in,  7,  161-165. 

Crab  blood,  pigment  of,   161,  162. 

Crab  eggs,  vitellolutein  and  vitello- 
rubin  in,  162. 

Crampton-Simons  palm-oil  test,  a  test 
for  carotinoids,  79,  220. 

Crataegus  crus-galli  (Cockspur  thorn), 
lutein  in  fruit  of,  75;  Crataegus  pin- 
natifida,  58. 

Crayfish,  carotinoids  of,  161,  162,  164, 
165. 

Cress,  garden,  pigment  of  etiolated 
leaves  of,  52. 

Cribella  oculata,  166. 

Crinoids,  165,  167. 

Crypt  onemiales,  101. 

Crocin,  22. 

Crocitin,  22. 

Crocus  sativus,  22,  74. 

Croton  ovaljolius  Vahl.,  microchemical 
crystals  in  yellow  spotted  leaves  of, 
55. 

Crown  Imperial,  carotinoids  in,  73. 

Crustacea,  carotinoids  in,  161-165,  171, 
179,  180,  181 ;  experiments  on  influ- 
ence of  color  of  food  and  surround- 
ings on,  189;  non-carotinoids  in,  154. 

Crustaceorubin,  23,   161,    165,   186. 

Cryptogams,  carotinoids  in,  92-124. 

Cryptomeria,  58. 

Cucumis  citrullis,  78 ;  Cucumis  melo,  76. 

Cucurbita  foetissima,  73;,  Cucuxrhita 
melanosperma  A.  Br.,  73;  Cucurbita 
pepo,  36,  78. 

Cup  fungi,   carotinoids  in,  116-118. 

Cup  Plant,  carotinoids  in  flowers  of, 
74. 

Cupressus  Naitnocki,  59. 

Cutleira  multifida,  96. 

Cyanophyceae,  110-112. 

Cyanophyll,  32,  34 ;  yellow  pigment  ac- 
companying, 37,  54. 

Cyclosporales,  95. 

Cymatopleura  solea,  107. 

Cymbyrhinchus  makrorhynchus,  144. 

Cyphomandra  betacea,  75. 

Cypress,  autumn  pigments  of,  59;  bald 
cypress,  58. 

Cyprinus  auratits,  147;  C.  Carpio,  147. 

Cypripedium  Boxolii,  C.  insigne,  C.  ar- 
gus,  74. 

Cystoseira  abrotanijolia,  95. 

Cyspoclonium  purpurascens,  101. 

Cytisus  laburnum  L.,  72,  73;  Cytisus 
sagittalis  Koch,  73. 


Dab,  carotin  in  skin  of  the,  147. 

Dacromyces  stillatv^,  114,  115. 

Daucus  carota,  18,  25;  variety  Bois- 
sieri  Schweinjurth,  anthocyanins  in, 
28. 

Daffodil,  carotin  in,  72;  yellow  pigment 
of,  7;  pigments  in  etiolated  leaves 
of,  52,  53;  xanthophylls  in,  73. 

Dagger-stab  pigeon  of  Luzon,  zoonery- 
thrine  in  red  feathers  of,  144. 

Dandelion,  69,  70;  carotin  in,  72;  xan- 
thophylls in,  73,  74. 

Dandruff  of  horse,  carotin  in,  135. 

Datura  stramonium,  249. 

Dellesseria  sanguinea,  101. 

Dendrobium  thrysiflorum  Rchb.  fil.,  72. 

Desmarestia  aculeata,  95. 

Diabetes,  carotinoids  in  blood  in,  207; 
excessive  skin  coloration  following 
carotinoid-rich  diet,  136,  151 ;  ex- 
planation of,  137,  151. 

Diaptoma,  18. 

Diaptomin,  23,  162. 

Diaptomus  bacillijer,  162,  183;  Diapto- 
mus  Castor  Jurine,  183. 

Diatoniaceae  (diatoms),  carotinoids  in, 
106-108,  122. 

Diatomin,  107,  108,  109,  183. 

Di-biphenylenathene,  a  non-carotinoid 
hydrocarbon  pigment,  21,  235. 

Di-carotin,  159,  177. 

Dictyopteris  polypodioides,  96. 

Dictyota  dichotoma,  96. 

Digestion,  effect  of  on  carotinoids,  196. 

DinoflageUata,  108. 

Dinophysis  acuta,  108;  D.  leavis,  108. 

Dioscora  batatas  decn.,  58. 

Discomycetes,  116,  117,  123. 

Ditiola  radicata,  114. 

Dog  Rose,  lycopin  in  fruit  of,  80. 

Doronicum,  pardalianthes  L.,  72;  Do- 
ronicum  Columnae  Tenore,  73; 
Doronicum  plantagineum  L.  excel- 
sum,  73. 

Doronium  Pardalianches,  73. 

Douce-amere,  76. 

Dryocapus  auratvs,  145. 

Dove,  lipochrome  in  blood  serum  of, 
140;  pigment  in  feet,  7. 

Dumontia  filijormis,  101. 

Dyer's  Greenwood,  carotinoids  in,  73. 

Dyer's  Woad,  carotin  and  xanthophylls 
in  flowers  of,  72. 

D.  Vinula,  157, 

Echinastrine,  167. 

Echinoderms,    carotinoids    in,    165-167, 

172. 
Echinoids,  165,  167. 
Eclectv^  polychlorus,  144,  145. 


306 


INDEX  OF  SUBJECTS 


Ectocarpaceae,  95. 

Egg    production,    correlation    of    with 

skin  colors,  272,  273,  277;   cause  of, 

273. 
Eggs,  carotinoid-free,  fertility  of,  268; 

vitamin  content  of,  270;  color  of  as 

affected  by  carrots,  271. 
Eggs,  xanthophyll  and  vitamin  content 

of  after  carrot  feeding,  271. 
Egg  yolk,  carotin-like  pigment  in,  139; 

color  of  affected  by  cayenne  pepper, 

187,  197;  pigment  of,  14,  15,  126,  127, 

138-140,    151,    173-177;    pigment    of 

carotinoid-free,  23. 
Egg  yolk  pigment,  as  cholesterol  ester 

of  oleic  acid,  139,  176;  solubility  of 

in  bile,  138. 
Egg    yolk    xanthophyll,    isomerism    of 

with    plant    xanthophyll,    176,    180; 

melting  point  of  affected  by  method 

of  determination,  179. 
Elachista  species,  95. 
Elaaeagnus  latijolia  L.,  54. 
Elaphis   quadrilineatis  Bonaparte,   150. 
Elder  leaves,  yellow,  carotinoids  in,  54. 
Elecampane,  carotinoids  in,  73. 
Elm,  autumn  pigments  of,  58,  59. 
Eloeis  guineesis  L.  (Jacq.),  77;  E.  Mel- 

anococca  Garb.,  77. 
Embryos,  mummified,  haematoidin  in, 

23. 
Encephalartos  Hildehrandtii,  59. 
English  Ivy,  carotin  in  leaves  of,  36; 

autumn  pigments  of,  58,  65. 
English   walnut,   autumn  pigments   of, 

58. 
Enterochlorophyll,  167. 
Enteromorpha  intestinalis,  104. 
Ephyra  Angularia,   157;   E.  Punctaria, 

157. 
Epimedium  macrantheum,  T6. 
Equestuni  arvense,  65. 
Eranthis  hyemalis  Salisb.,  73. 
Erysimum    Perojskianum    Fisch.    and 

Mey.,  73. 
Erythrophyll,    relation    of    to    carotin, 

34,  36,  37;  in  autumn  leaves,  56,  60. 
Erythroxylum,  nova-granadense,  77. 
Eschscholtzia  calijornica,  68,  74. 
Eteolin,  a  flavone,  21. 
Ethereal    oils,    as    solvent    for    chloro- 
phyll in  Kraus  separation,  32. 
Etiolated  leaves,  carotinoids  of,  48-53, 

89;  effect  of  greening  on,  53;  condi- 
tions  favorable   for  development   of 

carotinoids  in,  53. 
Etiolin,  absence  of  from  autumn  leaves, 

59;  relation  of  to  carotin,  49,  52. 
Eucarotin,  19.  105,  107,  177;  relation  of 

to  carotin,  19,  159. 


Eugenia  uniflora,  76. 

Euglena  sanguinea,   109,   110;   Euglena 

viridis,   109. 
Eunotia      (Himanthidium)     pectinalis, 

107. 
Euonymous  europaeus,  76;  Euonymous 

japoniciis  L.,   variety   sulphurea,   54, 

86;  E.  latifolia,  8Q. 
Euphone  nigricollis,  145. 
European  cranberry,  carotinoids  in,  77. 
European   elder,  carotinoids  in  yellow 

leaves  of,  54. 
Euxanthone,  21. 
Eye-spots  of  Euglena,  pigments  in,  109, 

110,  123. 

Fagus,  59,  60;   Fagus  silvatica,  255. 
Fatty    acids,    color    of    after    staining 

with  Nile  blue,  245. 
Fat  stains,  effect  of  on  carotinoids  in 

animal  tissues,  242,  244-246. 
Feathers,   artificial    coloration   of   with 

cayenne  pepper,  187,  197. 
Feathers,  carotinoids  of,   142-145,   151; 

origin  of  blue  color  of,  142. 
Fern  leaves,  carotin  content  of,  249. 
Ferric  chloride  reaction  of  carotinoids, 

26,  219,  222;  value  of  in  identifying 

carotinoids    in    animal    tissues,    247, 

248. 
Ferula  species,  73. 
Fir  leaves,  etiolated  pigments  of,  52; 

autumn  pigments  of,  65. 
Fish  carotinoids,  origin  of  from  food, 

184. 
Fish  colors,  influence  of  color  of  sur- 
roundings on,  183. 
Fishes,   pigments   in   skin    of,    145-148, 

152;    skin    pigments   and    chromato- 

phore  control  of,  146,  152;  structural 

blues  and  whites  of,  156. 
Flagellata,  109,  110. 
Flamingo,  pigments  in  feathers  of,  143, 

144. 
Flounder,  carotin  in  skin  of,  145;  effect 

of  light  on  colorless  side  of,  148. 
Flavones,  21,  55;  as  cause  of  autumn 

colors,  63;  in  flowers,  67. 
Flaxseed,  pigment  in  oil  not  carotinoid, 

87. 
Flowering  maple,  carotinoids  in  natu- 
rally yellow  leaves  of,  54;  carotin  in 

flowers  of,  72,  73. 
Flowers  of  Tan,  119. 
Flustra  joliacea,   168. 
Formic  acid  as  solvent  for  xanthophyll, 

225;  for  rhodoxanthin,  229. 
Forsythia  Fortunei,  73;  Forsythei  viri- 

dissma,  73. 
Four-o '-clock,  autumn  pigments  of,  58. 


INDEX  OF  SUBJECTS 


307 


Fowls,  xanthophyll  in  blood  serum  of, 
140;  extraction  of  xanthophyll  from 
blood  serum  of  with  ether,  214-. 

Fragilaria   species,   107. 

Fraxinus  excelsior,  58,  60. 

Frogs,  carotinoids  in,  149,  153;  loss  of 
skin  lipochromes  of  on  fasting,  185. 

Fringilla  canaria,  145. 

FritiLlaria  Iniperialis,  73. 

Fucoidiae,  94,  95. 

Fucoxanthin,  94-97;  amount  of  in  cer- 
tam  plants,  99,  257;  effect  of  alkalis 
on,  231,  232;  function  of  in  brown 
algae,  265;  halogen  derivatives  of, 
240;  in  blue-green  algae,  112;  in  red 
algae,  102;  methods  of  isolation  of, 
215,  216;  oxidation  products  of,  239; 
oxonium  salts  of,  98,  99;  relative 
solubility  properties  of,  98,  231,  257; 
special  properties  of,  97-99. 

Fucoxanthin  crystals,  color  and  form 
of,  239;  color  reactions  of,  240;  solu- 
bility of,  239. 

Fucoxanthin  solutions,  relative  color  of 
compared  to  carotin  and  xantho- 
phyll, 230;  compared  to  standard 
potassium  dichromate,  257. 

Fucoxanthophyll  in  brown  algae,  97; 
in  diatoms,  107;  in  dinoflagellates, 
109. 

Fucus  nodosus,  95;  F.  serratus,  95;  F. 
versoides,  95;  F.  vesculosus,  95. 

Fuligo  Septica,  119. 

Fulvenes,  as  non-carotinoid  hydrocar- 
bon pigments,  21,  235. 

Function  of  carotinoids,  in  plants,  263- 
265;  in  animals,  267,  268. 

Fungi,  carotinoids  in,  113-119. 

Funkia  Sieboldii,  58. 

Furcellaria  jastigiata,  101. 

Gaillarvia  splendens,  73. 

Gall  stones  of  cattle,  carotin  in,  182. 

Gasteromycetes,  113. 

Gasterosteus  spinachia,  147. 

Gazania  splendens  Hort.,  72,  73. 

Gebbia  littoralis,  165. 

Geese,  pigment  in  fatty  tissues,  shanks 

and  skin  of,  140,  145;  in  feet  of,  7, 

145. 
Genista  racenosa,  73;  Genista  tictoria, 

73. 
Gentisein,  21. 
Geum     montanum,    73;     Geum     coc- 

cineum,  74. 
Giant  Fennel,  carotinoids  in,  73. 
Gigartinales,  101. 
Ginkgo  biloba,  58. 
Ginseng,  autumn  pigments  of,  58. 
Gleditsia  triacanthos,  58,  62. 


Glenodinum  species,  108. 
Globe  flower,  carotin  in,  72,  74. 
Glycerin,    as   protective    agent   against 

oxidation  of  carotin,  206. 
Goat's  milk,  influence  of  feeding  carrots 

on  color  of,  189. 
Golden  Bell,  carotinoids  in,  73. 
Golden-tuft,  carotinoids  in  flowers  of, 

73. 
Goldfish,  lycopin-like  pigment  in  skin 

of,  147,  152;  zoonerythrine  in,  147. 
Gomphonema  species,   107. 
Gongora  galeata  Reichb.,  73. 
Goniaster  equestris,  166. 
Gonocarium  obovatum  Hocr.,  77;  Gon. 

pyrijorme  Scheff.,  77. 
Gooseberry,    pigment    of    red    autumn 

leaves,  56. 
Gorgonia  verrucosa,  169. 
Gorse,  xanthophylls  in  flowers  of,  73. 
Gossypium  hirsutum,  87. 
Gossyptin,  21. 
Grantia  coriacea,  170. 
Grains,  carotinoids  in,  86-88. 
Grape,  autumn  pigments  of,  58. 
Grape  leaves,  carotin  content  of,  249. 
Grass,  carotin  content  of,  249. 
Graptophyllum    pictum    Griff.,    micro- 
chemical   crystals   in  yellow   spotted 

leaves,  55. 
Grasshoppers,  160,  171. 
Great  red  macaw,  pigments  in  feathers 

of,  143,  145. 
Green    algae,    carotinoids    in,    102-106, 

122. 
Grenilabrus  melops,  161. 
Groundsel,  carotinoids  in  flowers  of,  74. 
Guernsey  cattle,  relation  of  skin  color 

to  fat  production  in,  275-277. 
Gymnodinium  Helix,  108. 
Gymnosporangium  juniperinum,  114. 

Haematochrome,  104,  105,  109. 
Haeinotococcus  pluvialis,  92,  103,   105, 

109,  117. 
Halma  Bucklandi,  170. 
Halichondria  albescens,  170;  H.  carwrv- 

cula,  170;  H.  incrustans,  170;  H.pani- 

cea,  170;  H.  rosea.  170;  H.  sanguinea, 

170;  H.  seriata,  170. 
Halidrys  siloquosa,  95. 
Halyseris  polypodioides,  96. 
Haematoidin    of   blood    exudates,   etc., 

23;  corpus  luteum,  14,  126. 
Haemolutein  of  corpus  luteum,  14,  126. 
Haemolymph  of  insects,  carotinoids  in, 

155-158. 
Haemolymph    xanthophyll,    origin    of 

from  food  in  case  of  caterpillars,  183, 

185,  197. 


308 


INDEX  OF  SUBJECTS 


Hawkbit,  crystalline  carotin  from,  69. 
Hawkweed,   carotinoids  in  flowers   of, 

73. 
Hawthorn,  autumn  pigments  of,  58. 
Hazelnut,  autumn  pigments  of,  58. 
Hedera  helix,  36,  58,  65,  249. 
Helenium  autumnale,  73. 
Helianthus  annus,  52,  67,  87. 
Helix  pomatia,  167. 
Hemerocallis  Middendirffii  Trautv.  and 

Mey.,   73. 
Hemp  leaves,  carotin  content  of,  249; 

etiolated  pigments  of,  52. 
Hemp  seeds,  effect  of  on  color  of  egg 

yolk,  87. 
Hens,  laying,  effect  of  feeding  red  pep- 
per to,  188. 
H  eterosithonales,  104. 
Hieracium  murorum  L.,  73;  Hieracium 

Pilosella,  73. 
Hircinia  spinosula,  170. 
Hippolyte  varians,  161,  189. 
Holothuria  nigra,  166;  H.  Ocnius  brun- 

neus,  166;  H.  Poli,  166;  H.  tuhulosa, 

166. 
Holothuroids,  165,  166. 
Honey  locust,  autumn  pigments  of,  58, 

62. 
Honeysuckle,    carotinoids   in   fruit    of, 

75,  80. 
Hop  tree,  autumn  pigments  of,  58. 
Hordeum  vulgare,  52,  88. 
Hornbeam,  European,  autumn  pigments 

of,  58. 
Horsetail,  red  carotinoid  in,  65. 
Hydroidictyon  utriculatum,  105. 
Hydrusus  penicillatus,  109. 
Hyla  arborea,  149. 
Hymeniacidon  albescens,  170. 
Hymenomycetes,  113,  114. 
Hyssopus  officinalis,  251. 

Identification  of  carotinoids  in  biologi- 
cal products,  240-247. 

Idotea,  influence  of  food  on  color  of, 
185. 

Imperfect  fungi,  carotinoids  in,  119. 

Indian  crocus,  pigment  of,  22. 

Iodine  derivative  of  carotin,  26,  27,  45. 

Iodine  color  reaction  of  carotinoids, 
limitations  of  for  animal  tissues,  243. 

Impatiens  Noli-tangere,  73. 

Insects,  pigments  of,  155-161. 

Internal  organs  of  mammals,  carotin- 
oids in,  133,  134. 

Inula  Helenium  L.,  73. 

Invertebrates,  carotinoids  in,  154-172. 

Iris  Germinia,  58;  Iris  pseudacorus  L., 
73. 

I  satis  tinctoria  L.,  72. 


Ithaginus  cruentatus,  144. 

Ivy  leaves,  carotin  content  of,  249. 

Japanese  Aokiba,  carotinoids  in  natu- 
rally yellow  leaves  of,  54. 

Japanese  Rose,  carotinoids  in,  74. 

Japanese  spindle  tree,  carotinoids  in 
naturally  yellow  leaves  of,  54;  in  red 
arils  of,  86. 

Jersey  cattle,  relation  of  skin  color  to 
fat  production  in,  275-277. 

Jerusalem  Cherry,  carotinoids  in,  75. 

Johannis   berries,   erythrophyll    of,   34. 

John  Dorey,  xanthophyll  in  skin  of,  147. 

Juglans  regia,  58,  249. 

Juniperus  virginiaca,  59. 

Kerria  japonica  D.  C,  74. 

Kidney    bean,     pigment    in    etiolated 

leaves  of,  50,  53. 
Kleinia  Galpini,  74. 
Kniphofia  aloodes,  74. 

Lacerta  agilis,  150;  L.  muralis,  150. 

Lacertofulvine,  relation  of  to  xantho- 
phyll,  150. 

Lactochrome,  131. 

Ladanum  hybridum,  74. 

Lady-slipper,  xanthophyll  in,  72;  other 
pigments  in,  74. 

Laminaria  saccharina,  93,  94,  96;  La- 
minaria  digitalis,  96. 

Lamium  album,  44. 

Larch,  European,  autumn  pigments  of, 
58. 

Larix  europaea,  58. 

Lark's  Spur,  cai'otinoids  in,  73. 

Laurencia  pinnatifida,  101. 

Leander  serrator,  161. 

Leathesia  marina,  96. 

Leaves,  carotinoid  content  of  as  af- 
fected by  surflight  and  shadow,  255. 

Lemania  fluviatilus,   101. 

Lemon,  absence  of  carotinoids  from,  78. 

Leontedon  autumnalis,  69,  74;  Leon- 
tedon  taraxacum,  74. 

Leopard's  bane,  xanthophyll  in,  72, 
73. 

Leotia  lubrica,  116,  117. 

Lepidium  Draba,  58;  Lepidium  sati- 
vum, 52. 

Lepidoptera,   155-158. 

Lepralia  foliacea,  168. 

Leucocyan  reaction  of  alcoholic  ex- 
ti'acts  from  brown  sea-weeds,  96; 
from  diatoms,  107. 

Leuconia  Gossei,  170. 

Leucoplastids,  29,  48. 

Leukophyll   in  etiolated   leaves,  49. 

Lichnoxanthin  in  fungi,  114. 


INDEX  OF  SUBJECTS 


309 


Liesegang  phenomena,  similarity  of 
Tswett's  chromatographic  analysis  to, 
43. 

Ligustrum  vulgar e,  65. 

Lilium  croceum  Chaix.,  72;  Lilium  bul- 
bijerum,  74. 

Lily-of-the-valley,  autumn  pigments  of, 
58. 

Lina-carotin,  23,  159. 

Lina  populi,  159;  L.  tremulae,  159. 

Linden,  autumn-xanthin  in  autumn 
leaves  of,  60;  carotin  content  of 
leaves  of,  249. 

Linium  lisitatissimum,  87, 

Lipochrin,  149. 

Lipochrome,  general  properties  of,  16; 
origin  of  name  of,  16;  pronunciation 
of,  9. 

Lipochromemia,  194. 

Lipochromogens  in  Crustacea  shells, 
transformation  of  into  lipochromes, 
164,  165,  172;  in  echinoderms,  1Q7, 
172. 

Lipocyan  crystals  produced  in  bacteria, 
121;  in  fungi,  114,  118,  119;  for  lina- 
carotin,  159. 

Lipoids,  effect  of  on  properties  of  ani- 
mal carotinoids,  13. 

Lipoxanthins,  general  properties  of,  17; 
origin  of  name  and  pigments  included 
among,  17. 

Liriodendron  tulipifera,  58,  74. 

Liver  of  arthropods,  cholechrome  in, 
168;  of  fish,  zoonerythrine  in,  148; 
of  mammals,  carotinoids  in,  134;  of 
molluscs,  cholechrome,  enterochloro- 
phyll  and  lutein  in,  167,  172;  of  mol- 
luscs, influence  of  food  and  hiber- 
nation on  pigments  in,  186. 

Lizards,  carotinoids  in,  149,  150;  chro- 
matophor  control  of  skin  color  in, 
150. 

Loasa  laterida,  74. 

Lobster  blood,  absence  of  tetronery- 
thrine  from  salt  water  species,  162; 
ether-soluble  pigment  in,  161. 

Locusta  viridissima,  160. 

Locusts,  160,  171. 

Lolium  perenne,  249. 

Lombardy  poplar,  autumn  leaf  pig- 
ments, 55. 

Loniceria  tataria,  75;  Lonciera  Xylos- 
teum,  80. 

Luff  a  gigantia,  251. 

Lutein,  as  specific  name  for  egg  yolk 
pigment,  15;  general  properties  of, 
15 ;  in  flowers,  67 ;  origin  of  name,  14. 

Lutechaematoidin  of  corpus  luteum,  14, 
126. 

Luteolin,  21. 


Luvarus  imperialis,  146. 

Lycaste  aromatica,  74. 

Lycogala  epidendron,  119;  L.  fiavofus- 
cum,  119. 

Lycopersicin,  83. 

Lycopersicum  esculentum,  69,  82. 

Lycopin,  as  cause  of  winter  color  of 
confiers,  66,  90;  composition  and 
properties  of,  83-85,  222-224;  difficulty 
of  separation  of  from  carotin,  224; 
effect  of  oxygen  on,  238;  factors  in- 
fluencing its  formation  in  tomatoes, 
266;  halogen  derivatives  of,  239; 
method  of  isolation  of,  215;  micro- 
chemical  identification  of  in  plant 
tissues,  241 ;  odor  of  during  oxidation, 
239. 

Lycopinoids  of  Lubimenko,  20. 

Lycopin  crystals,  color  and  form  of, 
238;  solubility  of,  238. 

L.  xanthophyll,  40,  42;  in  autumn 
leaves,  61;  relation  of  to  egg  yolk 
pigment,  173. 

Mackerel,  xanthophyll  in  skin  of  the, 

147. 
Maclaurin,  21,  57. 
Madura  aurantiaca,  57,  58. 
Macrozamia  species,  59. 
Maidenhair  tree,  autumn  pigments  of, 

58. 
Maize,   yellow,   carotinoids  in,   15,  87, 

91;  vitamin  A  in,  269. 
Maja  squinado,  18,  162. 
Manettia  bicolor  Taxt.,  72. 
Maple,  carotin  content  of  leaves,  249; 

common  European,  autumn  pigments 

of,  58;  Norway,  autumn  pigments  of, 

58. 
Marguerite,  carotinoids  in,  73. 
Marigold   flower,  carotin  and   xantho- 

phylls  in,  69,  73,  74. 
Maslevallia  Veitchiana,  75. 
Meconopsis  cambria  Vig.,  74. 
Medicago  sativa,  45. 
Megaloprepia  magnifica,  144. 
Meiampsora  Salicis  capreae,   114;   M. 

aecidiodes  D.  C,  114. 
Melasoma  populi,  159;   M.  XX-punc- 

tatum,  159. 
Melilopus  officinalis,  74. 
Merry  Sole,  carotin  in  skin  of  the,  147. 
Methyl-ethyl    maleic    acid    anhydride, 

relation    of    derivative    of    to    lipo- 
chrome, 236. 
Microchemical  crystallization  methods, 

28,  105,  241. 
Micrococcus  apatelus,  121;  M.  aureus, 

120,  121;  M.  Erythromyxa,  121,  122; 

Micrococcus    (Staph.)    pyogenes   av/- 


310 


INDEX  OF  SUBJECTS 


reus,  120;  M.  rhodochrous,  121;  M. 

superbiis,  121. 
Microcystis     (Polycystis)     flos    aquae 

Wittr.,  Ill,  112. 
Mildews,   carotinoids  in,    118. 
Milk  fat,  carotinoids  in,  131,  132,  151; 

human,  origin  of  color  of,  193. 
Milk  fat  formation,  significance  of  caro- 
tin-albumin complex  of  blood  in,  209. 
Milkweed,  carotin  in  flowers  of,  72. 
Milk  whey,  lactochrome  in,  131. 
Mimulus  mosthatv^  L.,  72. 
Miradilis  Jalapa,  58. 
Mirbius  viridis,  160. 
Mite,  common  red,  lipochrome  in,  161. 
Molds,  carotinoids  in,  118. 
Molisch  microchemical  technic  for  caro- 
tinoids, 241. 
Molluscs,  carotinoids  in,  167,  168. 
Momordica  balsamina,  72,  86;  Momor- 

dica  charantia,  76. 
Monilia  sitophila   (Mont.)   Dace,  119. 
Mono-carotin,  159,  177. 
Morin,  57. 
Morus  alba,  36,  58. 
Mouse-ear    Hawkweed,    carotinoids   in 

flowers  of,  73. 
Mucor  flavus  Bainer,  118. 
Mulberry,    autumn    pigments    of,    58; 

leaves,  carotin  in,  36. 
Mullein  pollen,  carotin  sole  pigment  of, 

71,  115. 
Mullein,    xanthophylls    in    flowers    of, 

73,  74. 
Mullus  barbatus,  147. 
Muraena  Helena,  147. 
Mushrooms,   carotinoids  in,   113,   114. 
Muskmelon,  pigments  in  fruit  of,  76. 
Musk    plant,    xanthophylls    in    flowers 

of,  72. 
Mussels,  carotinoids  in  liver  of,  167. 
Mustard-seed  oil,  pigment  of  not  caro- 

tinoid,  87. 
Mycetozoa,  119. 
Myristica  jragrans   Houtt.,  87. 
Mytilv^  edulis,   167. 
Myxomycetes,  118,  119. 
Myxophyceae,  110-112. 

Nasturtium,  xanthophylls  in,  73,  74; 
other  pigments  in,  75. 

Narcissus,  poet's,  carotin  the  predomi- 
nating pigment  of  carona  of,  71,  72. 

Narcissus  poeticu^  L.,  72;  Narcissus 
Pseudo-narcissus  L.,  72,  73;  Narcis- 
sus iaxetta,  75. 

Narcissus  Polyanthus,  anthocyanins  in, 
75. 

Naturally  yellow  leaves,  carotinoids  in, 
53-55. 


Navicula  viridis,  107. 

Necrobiotic  phase  of  autumn  colora- 
tions, 56. 

Neciria  cinnabarina,  116,  117. 

Nectriin,  or  nectria  red,  117,  118. 

Nemalionales,  101. 

Nenga  Schejferiana  Becc,  77. 

Nephrops  norwegicus,  164. 

Nertera  depressa  Banks  and  Soland,  77. 

Nereis  virens,  168. 

Nerophis  oequoreus,  147. 

Nerves,  carotinoids  in,  134,  135. 

Nettle  leaves,  carotin  from,  36,  45; 
dead  nettle,  carotin  and  xanthophyll 
in,  44. 

Neutral  fats,  color  of  after  staining 
with  Nile  blue,  245. 

Nodularia,  111. 

Nomenclature,  causes  of  diversity  of 
among  yellow  animal  pigments,  13; 
among  yellow  vegetable  pigments,  14. 

Non-carotinoids,  yellow  and  orange 
colored  in  animals,  22,  in  plants,  21. 

Nonnea  lutea  D.  C,  72. 

Nostocacea,  111. 

Nicotinia  tabacum,  249. 

Nile  blue,  differentiation  of  neutral  fats 
and  fatty  acids  by,  245;  effect  of  on 
animal  carotinoids,  245,  246. 

Nitella  spores,  102. 

Nitzschia  Palea,  107;  Nitzschia  sig- 
moidea,  107. 

Nuphar  luteum  Sibth.,  74. 

Nutmeg,  non-carotinoid  pigments  in 
aril  of,  87. 

Nyctanthin,  a  yellow  pigment  re- 
sembling carotinoids  in  composition, 
22. 

Oak,  English,  autumn  pigments  of,  58; 
red,  autumn  pigments  of,  58,  60. 

Oat  leaves,  etiolated  pigments  of,  52, 
53;  oat  grain,  carotinoids  in,  88. 

Odintiglossums,  75. 

Odor  of  oxidizing  carotin,  233;  lycopin, 
239;  xanthophyll,  237. 

Oedogoniales,  104. 

Oenothera  biennis,  74. 

Olea  Europaea,  249. 

Oleander  leaves,  autumn  yellow  pig- 
ments of,  59. 

Olive  leaves,  carotin  content  of,  249. 

Oncidiums,  75. 

Onion  flowers,  pigments  of,  74. 

Ophiurine,   167. 

Ophiuroids,  165,  167. 

Orange  Hawkweed,  xanthophylls  in 
flowers  of,  72. 

Orange  skin,  carotinoids  and  other 
pigments  in,  81. 


INDEX  OF  SUBJECTS 


311 


Orangin,  an  orange  pigment  in  skin  and 

liver  of  starfish,  166. 
Orange  xanthophyll  of  Sorby,  in  brown 

algae,   95;    m  blue-green   algae,   112. 
Orchid,  xanthophylls  in,  72;   anthocy- 

anins  in,  75. 
Origin    of    animal    carotinoids,    early 

theories  regarding,  182,  183. 
Origin  of  plant  carotinoids,  266. 
Orthoptera,   160. 
Oryza  sativa,  88. 

Osage  orange,  pigments  in  yellow  au- 
tumn leaves  of,  57,  58. 
Oscillatoria  iOscillaria)  limosa,  111;  0. 

laptotricha,    111;    0.   Froelichii,   111. 
Osmerus  eperlanus,  147. 
Osmic  acid,  as  dye  for  carotinoids,  244. 
Oxidation,    effect    of   on   properties    of 

carotinoids,  13,  14,  27,  218,  231,  233, 

238,  246. 
Oxidation    of    carotinoid    granules    in 

animal  tissues,  effect  of  on  staining 

properties,  246. 
Oxidase  in  animals,  as  cause  of  lack  of 

carotinoids,  196. 
Oxonium  salts  of  fucoxanthin,  98,  99, 

231. 

Pachymatisvia  Johnstonia,  170. 

Padina  Pavonia,  96. 

Palm    oil,    carotinoids    of,    77;    Palm 

fruits,  carotinoids  of,  77. 
Paloemon  viridis,  165. 
Pandanus  polycephalus   Lam.,   77. 
Pansy,  xanthophylls  in,  73. 
Paradisea  papuana,  143,  145;  P.  rubra, 

143. 
Paradiseofulvine,  144. 
Paroaria  cucuUata,  144. 
Parrots,    red    and   yellow   pigments    in 

feathers  of,  143-145. 
Parsley,  crj'-stalline  carotinoids  from  by 

Borodin,  35. 
Paxsnip  root   (Pastinaca  sativa),  28. 
Passijlora  coerulea,   86. 
Passion  flower  plant,  pigment  in  arils 

of,  86. 
Papilio  Machaon,  157. 
Papillina  suberea,  170. 
Peach  tree  leaves,  carotin  in,  36;  caro- 
tin content  of,  249;  erythrophyll  of, 

34. 
Pea  leaves,  carotin  content  of,  249. 
Pear  tree,  pigment  of  autumn  leaves 

of,  31,  56,  58. 
Pepperwort,    autumn   pigments   of,  58. 
Perca  fluviatilis,  186. 
Perch,    crab-eating,    influence    of    food 

on  color  of,  186. 
Peridinieae,  108,  122. 


Peridinin,  108. 

Peridinium  divergens,  108. 

Persica  vulgaris,  36,  249. 

Petroleum   ether  in  Kraus  separation, 

32. 
Petroselinum  sativum,  35. 
Periwinkle   leaves,   carotin   content   of, 

249. 
Peziza  aurantia,   116,   117;    P.   bicolor, 

116;  P.  scutellata  L.,  116. 
Peziza  xanthin,   116. 
Phaeophyceae,     93-100;     quantity     of 

carotinoids  in,  99. 
Phaeosporales,  95. 
Phaseolus   vulgaris,  50,  249. 
Phenol-glycerine     reagent     in     Molish 

microchemical  crystallization  method, 

105,  118,  237,  242. 
Philobolus  crystallinus,  118;  P.  Kleinii, 

118;  P.  Oedipus,  118. 
Phlegoenus  cruenta,   144. 
Phormidium  vulgare.  111. 
Phragmidium  violaceum,  114. 
Phrrhocoris   apterus,    carotin    in    tegu- 
ment of,  158,  177. 
Phycoerythrin,    as    chief    pigment    in 

red  algae,    100,    122;    analogous   pig- 
ment in  Dinoflagellates,  108. 
Phycochrome,  111. 
Phycocyan,  96,  111. 
Phy  corny  cetes,   118. 
Phycopelpis  amboinumis,  105;  P.  aurea, 

105;  P.  epiphyton,  104;  P.  maritima, 

105;  P.  teudii,   105. 
Phycophain,  a  post-mortal  pigment  in 

brown  sea-weeds,  94,  96,  98,  122. 
Phycoxanthin,  94,  95,  97,  107,  111,  112. 
Phyllocyanine,  29,  49,  56. 
Phyllodium    dimorphum,    105 ;    P.    irv- 

certums,  105. 
Phyllofuscin,  54. 
Phyllospora  Brodiaei,  95;  P.  membrani- 

jolia,  95. 
Phylloxanthine,  29-31,  33,  49,  56,  60. 
Phylogenetic  origin  of  carotinoids,  126. 
Physalis  alkekenzi,  79;   P.  Franchetti, 

74,  80. 
Physico-chemical    properties    of    carof- 

tinoids,  43. 
Phytophagus     larvae,     carotinoids    in, 

155-161. 
Pyrochra  coccinea,  159. 
Picides,  144,  145. 
Picofulvine,  144. 
Picus  major,  144. 
Pigeons,   pigment   in  blood   serum   of, 

140;  on  legs,  195. 
Pipe  Fish,  carotin  and  xanthophyll  in 

skin  of,  147. 
Pisum  sativum,  52,  249. 


312 


INDEX  OF  SUBJECTS 


Plaice,  carotin  in  skin  of,  147. 

Plantago  (plaintain),  44. 

Plant  lice,  green,  carotinoids  in,  158, 
171. 

Plant  tissues,  microchemical  identifica- 
tion of  carotinoids  in,  240-242. 

Plaster  of  Paris,  as  aid  in  isolation  of 
carotin  from  blood,  207. 

Platanus  acerijolia,  255;  Platanus  ori- 
entalis,  68. 

Platisama  Cocropia,  157. 

Pleotrachelus  julgens,  118. 

Pleuronectes  flesus,  147;  P.  platessa, 
147;  P.  limanda,  147;  P.  microcepha- 
lus,  147. 

Poison  ivy,  autumn  pigments  of,  58. 

Polychaetes,  carotinoids   in,   168. 

Polychrome,  56. 

Polycystin  from  Polycystis  flos  aquae, 
relation  of  to  carotin,  112. 

Polygonum  sachalinense,  68. 

Polyides  rotundvs,  101. 

Polynoe  spinijera,  168. 

Polysiphonia  species,  101 ;  Polysiphonia 
nigrescens,  101. 

Polystigma  rubrum,  116,  117;  P.  Ochra- 
ceum  {P.  julvum  D.  C),  116,  117, 
118. 

Polystigmin,  properties  of,  117. 

Polyzoa,  lipoclirome  in  cuticular  skele- 
ton of,  168. 

Pond  Lily,  European,  carotinoids  in, 
74. 

Pond  weed,  rhodoxanthin  in,  47,  66,  216. 

Pontellina  gigantea,  164. 

Poplar,  autumn  pigments  of,  58,  60. 

Poppy,  Welsh,  carotinoids  in,  74. 

Populv^  alba,  58;  Populus  canadensis, 
255;  Populus  jastigiata,  55;  Populus 
nigra,  58;  Populus  tremula,  58. 

Porifera,   169. 

Porphyra  hiemalis,  101;  Porphyra  laci- 
niata,  101;  Porphyra  vulgaris,  100, 
101. 

Postmortal  phase  of  autumn  colora- 
tion, 56,  63. 

Potamogeton  natans,  47,  66,  216. 

Potassium  dichromate  standard,  color 
of  in  comparison  with  carotinoids, 
253,  254,  260. 

Potato  leaves,  carotin  content  of,  249. 

Potato  sprouts,  effect  of  light  and  dark 
on  carotinoids  in,  53. 

Potato,  sweet  (Ipomoea  batatas),  28. 

Prawns,    carotinoids    in,    161. 

Primrose,  European  Evening,  carotin- 
oids in,  74. 

Primula  officinalis,  72. 

Privet,  winter  reddening  of,  65. 

Promycelia   spores,   114,  116. 


Prorocentum  micans,  108. 
Protochlorphyll,  relation  of  to  etiolia, 

50. 
Protococcus    {Pleurococcus)    pluvialis, 

105;  P.  vulgaris,  105. 
Prunus  armeniaca,  76;  Prunus  avium, 

68;  Prunus  cerasus,  34,  56. 
Pseudozoorubin,   143. 
Psittacofulvine,    144. 
Ptelea  trijoliata,  58. 
Pteris  aquilina,  249. 
Ptychandra  glauca  Scheff.,  77. 
Ptychosperma  elegans  Blume,  77. 
Puccinia  coronata,  114. 
Puff-balls,  113. 

Pumpkin,  carotin  in,  36,  78,  79. 
Pygaera  Bucephalus,   156;   P.  meticvr 

losa,  156. 
Pylaiella  litoralis,  95. 
Pyrenomycetes,  116,  117,  123. 
Pyrocephalus  rubincus,  144. 
Pyrrhula  vulgaris,  144. 
Pyrus  communis,  29,  56,  58;  Pyrvs  ger- 

manica,  58;  Pyrus  ussuriensis,  58. 

Quercus  rubra,  58,  60;  Quercus  Rohur, 
58. 

Rana  esculenta,  149,  185. 

Ranunculus,  69;  Ranunculus  acris  L., 
73;  R.  Aricomus,  7i;  R.  Ficaria,  74; 
R.  gramineus,  74;  R.  repens,  74. 

Rape  leaves,  carotin  content  of,  249. 

Rape  seed,  effect  of  on  color  of  egg 
yolks,  87. 

Rape-seed  oil,  pigment  of  not  carotin- 
oid,  87. 

Raphanus  raphanistrum  L.,  73. 

Rats,  growth  of  on  carotinoid-free  diets, 
270. 

Red  Algae,   carotinoids  in,   100-102. 

Red  currant  leaves,  carotin  content  of, 
249. 

Red  pepper  pigment,  69,  82;  feeding 
of  to  canaries  and  fowls,  187. 

Red  xanthophyll,  20,  47. 

Reptiles,  carotinoids  in,  149,  150. 

Retina,  carotinoids  in,  126,  140-142, 
149,  152. 

Retinal  epithelium,  absence  of  col- 
ored globules  from  in  cow,  man,  pig, 
and  snakes,  142. 

Retinostora  plumosa,  59. 

Rhinescis  scalaris,  150. 

Rhodophane,  23;  in  bird  feathers,  143; 
relation  of  to  zoonerythrine,  143;  iu 
bird  retinas,  141-142;  relation  of  to 
tetronerythrine,  142;  in  crufetacea, 
162;  in  echinoderms,  166;  in  worms, 
168. 


INDEX  OF  SUBJECTS 


313 


Rhodophane-like  pigment  in  sponges, 
169;  in  Tubularia  indivisa,  169. 

Rhodophyceae,  100-102. 

Rhodoxanthin,  20,  47,  66,  90;  method 
of  isolation  of,  216,  217;  micro- 
chemical  identification  of  in  plant 
tissues,  241. 

Rhodoxantliin  solutions,  properties  of, 
229-230. 

Rhodymeniales,  101. 

Rhus  toxicodendron,  58. 

Rhynchota,  158. 

Ribes  aureum,  73;  Ribes  rubrum,  249. 

Ribus  glossularia,  variety  rubra,  56;  Ri- 
bus  nigrum,  34. 

Rice,  polished,  carotinoids  in,  88. 

Rivulariaceae,   111. 

Robinia  pseudo-acacia,  249. 

Rosa  canina,  80. 

Rosaceae,  autumn  reddening  of,  65. 

Rosa  rugosa,  58,  81;  Rosa  species,  74; 
pigments  in  fruits  of,  80. 

Roots,  yellow,  need  of  study  of  pig- 
ments in,  88. 

Rugosa  rose,  autumn  pigments  of,  58; 
xanthophylls  in  fruit  of,  81. 

Rubicen,  a  red  non-carotinoid  hydro- 
carbon, 236. 

Rubus  caesius,  251. 

Rudbeckia  Neumanii,  74. 

Rutabaga,  pigment  of,  29. 

Ruta  graveolens,  251. 

Rusts,  carotinoids  in,  113-116,  123; 
function  of  carotinoids  in,  263. 

Rye  straw,  yellow  pigment  of  in  au- 
tumn, 59. 

Sacaline,  autumn  pigments  of,  58. 
Salamanders,   carotinoids   of,   149,   153. 
Salamandra  maculosa,  149. 
Salix  babylonica,  58. 
Salix  Caprea,  58. 

Salmon,  fadmg  of  flesh  of  during  mi- 
gration to  spawning  beds,  274. 
Salmon  muscle,  pigments  of,  147,  148, 

152;  as  modified  carotinoid,  195. 
Salmon  pigments,  influence  of  food  on, 

184. 
Sambucus  nigra  L.,  variety  aurea,  54; 

Sambucus  nigra  joliis  luteis,  54,  55. 
Saponification,  effect  of  on  carotinoids, 

205. 
Saturnia  Pernyi,  157;  S.  Pyri,  157. 
Saxijraga  umbrosa,  winter  reddening  of, 

65. 
Scarlet  Red,  as  dye  for  carotinoids  in 

animal  tissues,  244. 
Scomber  scombrus,  147. 
Scorpoena  scroja,  147. 
Scotinosphaera  paradoxa,   105. 


Scytonemaceae,  111. 
Sea  cucumbers,  carotinoids  in,  165,  166. 
Sea  lihes,  carotinoids  in,  165,  167. 
Sea  urchins,  carotinoids  in,  165,  167. 
Seeds,  carotinoids  in,  86-88. 
Stlaginella,  59,  65. 
Selencides  alba,  145. 
Sesame-seed  oil,  pigment  of  not  a  caro- 
tinoid, 87. 
Sesamum  indicum,  87. 
Shanks,  pigments  of  skin   of  in  fowls 

and  other  birds,  140,  145. 
Silk,   color   of   as   influenced   by  color 

of  blood  of  silkworm,  186. 
Silphium  perjoliatum,  74. 
Silver  poplar,  autumn  pigments  of,  58. 
Sinapadix  Petrichiana  Hort.,  77. 
Sinapis  alba,  52,  74. 
Siphocampylos    bicolor   G.   Dow.,   72. 
Siphonales,  104. 
Siphonocladiales,  104. 
Siphonostoma     diplochaitos,    168;     Si- 

phonostoma  typhle,  147. 
Sisyvibriutn  Sophia,  74. 
Sittace  Macao,  143,  145. 
Skin,  cause  of  fading  of  in  fowls  during 

egg  laying,  273. 
Skm  of  mammals,  carotinoids  in,  135- 

137;   effect  of  diet  on,  135-137,  193, 

194,  273. 
Slime   molds,   carotinoids  in,   118,   119, 

123. 
Smelt,  carotin  in  skin  of,  147. 
Smirinthus  Oscellatus,   156;   S.  Populi, 

156;  S.  Tiliae,  156. 
Smuts,  113. 
Snails,    carotinoid   in   liver   and   shells 

of,  167,  172. 
Snakes,  carotinoids  in,  149,  150. 
Sneezewood,  carotinoids  in  flowers  of, 

73. 
Solanorubin,  82,  104. 
Solanuin   corymbosum,   76;    S.   decase- 

"palum,  77 ;   S.   dulcamara,  81 ;   Sola- 

num  pseudo-capsicum,,  75;   Solanum 

tuberosum,  249. 
Soiaster  papposa,  166. 
Solea  vanegaia,  147. 
Solvents  for  carotinoids,  204. 
Sorbus  aria,  Crantz.,  80;  Sorbus  aucu- 

paria,  56,  81. 
Spaerostiibe  coccaphila,  116,  118. 
Spaerotilus  roseus,  120,  121,  123. 
Sparmania  ajricana,  58. 
Spathularia  jlavida  Pers.,  116,  117. 
Specificity    of    carotinoids   in    animals, 

origin  of,  196,  197. 
Spectroscopic   absorption  properties  of 

carotin    solutions,    220-222;    of    solid 

carotin,  222;  of  fucoxanthin,  231;  of 


314 


INDEX  OF  SUBJECTS 


lycopin,  223,  224;  of  rhodoxanthin, 
230;  of  crystalline  xanthophyll,  223, 
228;  of  xanthophyll  a,  228;  of  xan- 
thophylls  a'  and  a' ',  229 ;  of  xan- 
thophyll 3,  229. 

Spermothamnion  roseolum,   101. 

Sphaerella  {HaemaiocciLS  or  Chlamy- 
dococcus)   pluvialis,  105. 

Sphaeropleacea,   104. 

Sphinx  Ligustru,  157. 

Spidei-  crab,  red  pigment  in  eggs  of, 
162,   163. 

Spinach,  as  source  of  carotin,  36;  caro- 
tin content  of,  249,  250. 

Spinachia  inermis,  249;  Spinachia 
oleracea  and  glabra,  36. 

Spindle-tree,  European,  pigments  in 
fruits  of,  76. 

Spiraea  species,  variety  aurea,  54. 

Spirogyra,  35,  103,  104;  Spirogyra  cras- 
sa,  104;  S.  maxima,  104. 

Sponges,  carotinoids  in,  169,  170,  172, 

Sporidia,  114,   116. 

Spring  Adonis,  carotinoids  in  flowers 
of,  73. 

Squash,  pigments  in  yellow  varieties  of, 
79. 

Startium  junceum,  L.,  73. 

Stemonitis  jerruginea,  119;  S.  jusca, 
119. 

Stichococcus  majus,  104. 

Stilophorum  diphyllum  Nutt.,  72. 

Stinging  nettle,  carotin  content  of,  249, 
250. 

Stone-worts,  carotinoids  in,  102,  122. 

Stramonium,  carotin  content  of,  249. 

Stredtzia  Regmae,  74. 

Strawberry  Tomato,  carotin  in,  79. 

Suberites  flavus,  170;  S.  massa,  170;  S. 
domuncula,  170. 

Sudan  III,  as  dye  for  carotinoids  in 
animal  tissues,  244;  effect  of  feeding 
to  fowls,  138,  274. 

Sudanophiles,  245. 

Sulphuric  acid  color  reaction,  non- 
specificity  of  for  carotinoids,  233, 
243. 

Sunflower  leaves,  etiolated  pigments  of, 
52. 

Sunflowers,  xanthin  in,  67;  xantho- 
phylls  in,  72. 

Sunflower-seed  oil,  pigment  of  not  a 
carotinoid,  87. 

Sycamore  leaves,  autumn  pigments  of, 
58;  carotin  content  of,  249;  carotin 
in,  36. 

Syngnathus  acus,  147. 

Tageies  erecta,  73;  Tagetes  patula,  74. 
Tamus  communis,  80,  81. 


Tanernomontana  pentastycha  Scheff., 
77. 

Taxodium  distichum,  58;  Taraxacum 
officinale,  70,  72,  73. 

Taxus  baccata,  59,  86,  216,  249. 

Tedania  Muggiana,  170. 

Telea  Polyphemus,  157. 

Telekia  speciosissima,  74. 

Terebella  species,  168. 

Tethya  Lyncureum,  170. 

Tetronerythrin,  23;  in  blood  of  lobster, 
crab  and  crayfish,  162;  in  fish  livers, 
148;  in  fish  skins,  146;  in  wattles  and 
"roses"  of  pheasants,  142. 

Thermopsis  lanceolata  R.  Br.,  74. 

Thuja  ericoides,  65;  Thuja  orientalis, 
59,  66,  216,  230,  251 ;  Thuja  standishi, 
65. 

Thuj  orhordin,  65,  66;  relation  of  to 
rhodoxanthin,  66. 

Tiga  tridactyla,  145. 

Tigerfinch,  pigments  in  feathers  of, 
143,   144. 

Ttlia,  60;  Tilia  platyphylla,  249. 

Tillandsia  splendens,  74. 

Toads,  carotinoids  of,  149,  153;  loss  of 
skin  colors  of  in  winter,  185. 

Toadstools,  carotinoids  in,  113,  114. 

Tobacco  leaves,  carotin  content  of,  249. 

Tolypothrix  species.  111. 

Tomato  pigment,  69,  75,  82-85;  tomato 
plastids,  68. 

Tomatoes,  carotin  and  lycopin  forma- 
tion in  during  ripening,  266;  effect  of 
temperature  of  ripemng  on  lycopin 
formation  in,  266;  isolation  of  ly- 
copin from,  215;  suppression  of  ly- 
copin in,  266. 

Torch  Lily,  carotinoids  in,  74. 

Tortoise,  xanthophyll  in,  150,  153. 

Torula  cinnabarina,  119;  T.  rubra,  119. 

Touch-me-not,  carotinoids,  73. 

Tree  tomato,  lutein  in,  75. 

Trentepholia  {Chroolepus)  aurea,  104; 
T.  aureum-tomentosum,  104;  T.  bi- 
sporangiata;  T.  crassiaetta;  T.  cy- 
ania;  T.  jolithus;  T.  maxima;  T. 
monilijormis;  T.  umbrina,  104, 

Tricosanthu^,  76. 

Trigla  cuculv^,  147;   T.  hirundo,  147. 

Tnticum  vulgare,  88,  249. 

Triton  cristatus,   149. 

Tritonia  aurea,  74. 

Trogon  Massera,  144. 

TroUivs  asiaticus  L.,  72;  Trollius  euro- 
paeus,  74, 

Trombidium,  161. 

Tropaeolum  majus,  73,  74 ;  T.  minus,  74. 

Tropical  fruits,  pigments  of,  76,  77. 

Tubularia  indivisa,  169. 


INDEX  OF  SUBJECTS 


315 


Tulipa   Gesncriana  L.,  73,  74;    Tulipa 

hortensis  Gagrtm.,  74. 
Tulips,    anthocyanins    in,    75;    xanthq- 

phylls  in,  73. 
Tulip   tree,   autumn  pigments  of,   58; 

carotin  in  flowers  of,  72. 
Turk's-cap  gourd,  pigments  of,  78. 
Turnip   leaves,   etiolated   pigments   of, 

52. 
Turnip  root,  yellow  (Brassica  Rapa  L.), 

relation  of  red  pigment  in  to  lycopin, 

29. 
Turtles,  carotinoids  in,  149,  150. 
Tussilago  Farjara  L.,  73. 

Uranidine,  a  skin  pigment  of  sea  cu- 
cumbers, 166. 

Uredineae,  113-116,  263. 

Uredo  euphrasix,  114,  116. 

Uredo  spores,  color  of,  115. 

Uric  acid  derivatives,  butterfly  pig- 
ments caused  by,  155. 

Uromyces  alchemillae,  114. 

Urtica,  36,  45;  Urtica  dioica,  as  source 
of  carotin,  202;  carotin  content  of, 
249. 

Ulex  europaeus,  73. 

Ulmus  campestris,  58,  59. 

Ulva  lactuca,  quantity  of  carotinoids 
in,  103. 

Ulvales,  104. 

Unsaponifiable  matter  of  fats,  caro- 
tinoids and  vitamin  A  in,  206. 

Uvalaria  grandiflora,  74. 

Vaucheria  species,  104. 

Vegetable  oils,  as  solvent  for  chloro- 
phyll in  Kraus  separation,  32;  lack 
of  correlation  between  pigmentation 
and  vitamin  content  of,  270. 

Verbascum  species,  73;  Verbascum 
thapsiforma,   71,   74,    115. 

Viburnum  Opulus,  77;  Viburnum  Tinus, 
251. 

Vinca  Major,  249. 

Viola  biflora,  74;  Viola  cornuta  L.,  va- 
riety Daldowie  yellow,  74;  Viola 
lutes,  74;  Viola  odorata,  71,  74,  249; 
Viola  tricolor  L.,  73. 

Violet  algae,  104,  105. 

Violet,  Horned,  carotinoids  in,  74; 
sweet,  quantity  of  carotin  in,  71,  247; 
carotinoids  in,  74;  yellow  petal,  caro- 
tinoids in,  7. 

Virbius  viridis,  164.  

Vitamin  A,  extractability  of  from  al- 
falfa, carrots  and  yellow  maize,  269; 
relationship  of  to  carotinoids,  268-270, 
271. 

Vitellolutein,  162. 


Vitellorubin,  23,  162. 

Vitis  coignetiae,  58;  Vitis  vinijcra,  249. 

Waldsteinia  geoides,  74. 
Wall-flower,   xanthophylls  in,   72;    an- 
thocyanins in,  75. 
Walnut  leaves,  carotin  content  of,  249. 
Waste  products,  carotinoids  as  in  plants 

and  animals,  262,  263. 
Watermelon,  pigments  of  flesh  of,  78. 
Wheat  leaves,  carotin  content  of,  249; 

flour,  unbleached,  carotinoids  of,  88; 

seedlings,   etiolated  pigments  of,  52. 
White  Beam-tree,  carotinoids  in  fruit 

of,  80. 
Wild  Ginger,  carotin  in  fruit  of,  79. 
Willow,  Goat,  autumn  pigments  of,  58; 

weeping,  autumn  pigments^  of,  58. 
Winter  Aconite,  carotinoids  in,  73. 
Woodpecker,  pigments  in  feathers  of, 

143-145. 
Worms,  carotinoids  in,  168,  169;  color 

of  as  influenced  by  food,  186;  non- 

carotinoids  in,  154. 
Wrasse,  carotinoids  in,  161. 

Xantheins,  relation  to  anthocyanins  and 
flavones,  67. 

Xanthemia,  136. 

Xanthia  flavago,  157. 

Xanthin  of  C.  Krause  in  autumn 
leaves,  relation  of  to  carotin,  60;  of 
Dippel,  relation  to  carotin  and  xan- 
thophyll,  34;  in  yellow  leaves,  54; 
of  Fremy  and  Cloez,  67.' 

Xanthocarotin,  38,  39;  alleged  transfor- 
mation of  to  xanthophyll,  39;  rela- 
tion of  to  carotinoids,  39. 

Xanthomelus  aureus,  144,  145. 

Xanthones,  21.  _ 

Xanthophanes  in  bird  retinas,  rela- 
tion of  to  xanthophyll,  142. 

Xanthophane-like  pigment  in  Tubu- 
laria  indivisa,  169. 

Xanthosis,  194. 

Xanthophyll,  absorption  of  oxygen  by, 
237,  238;  alleged  production  of  from 
carotin  and  chlorophyll,  265;  consti- 
tution of,  238;  effect  of  alkalis  on, 
225,  226;  excretion  of  by  skin  in 
fowls,  274;  halogen  derivatives  of, 
237;  isolation  of  from  blood  serum, 
214,  215;  from  egg  yolk,  212-214; 
from  green  leaves,  209-212,  251;  lack 
of  hydroxyl,  carboxyl  and  carbonyl 
groups  in  molecule  of,  47;  origin  of 
name  of,  31 ;  reduction  of  to  carotin, 
48;  relation  of  to  carotin,  24. 

Xanthophyll  a  of  Tswett;  distinguish- 
ing properties  of,  228;  relation  of  to 


316 


INDEX  OF  SUBJECTS 


crystalline  xanthophyll  of  Willstat- 
ter,  45;  to  egg  yolk  xanthophyll,  45, 
139. 

Xanthophyll  (3  of  Kohl,  not  true  xan- 
thophyll, 44. 

Xanthophyll  P  of  Tswett,  blue  color 
reaction  of  alcoholic  solution  of  with 
HCl,  33,  229;  relation  of  to  crystal- 
line xanthophyll,  45. 

Xanthophyll  in  brown  sea-weed,  96; 
in  red  sea  weeds,  102,  122. 

Xanthophyll  in  egg  yolk,  15,  138-140; 
composition  and  properties  of,  174; 
isomerism  of  with  plant  xanthophyll, 
176,  180;  method  of  isolation  of,  173, 
174,  209-215;  origin  of,  139,  197,  268; 
relation  of  to  xanthophyll  a,  45,  139, 
151;  variation  in  melting  point  of 
from  plant  xanthophyll,  175. 

Xanthophyll  crystals,  color  and  form 
of,  236;  color  reactions  of,  237;  odor 
of  during  oxidation,  237;  properties 
of,  224,  236-238;  solubility  of, 
237. 

Xanthophyll  series  of  pigments  ac- 
cording to  Kohl,  38,  141 ;  according 
to  Schunck,  38,  40,  41;  according  to 
Tschirch,  37,  38. 

Xanthophyll  solutions,  properties  of, 
224-229;  relative  color  of  compared 
to  carotin,  225. 

Xanthophylls,  effect  of  acids  on  alco- 
holic solutions  of,  40,  41,  228,  229; 
method  of  separation  of  by  chroma- 
tographic   analysis,    226-228;    micro- 


chemical  identification  of  in  plant 
tissues,  241,  242;  separation  of  from 
carotin,  252,  253. 

Xanthophylls  a'  and  a",  distinguish- 
ing properties  of,  228,  229. 

Xanthophylloids  of  Lubimenko,  20. 

Xanthophyll-rich  food,  effect  of  on 
color  of  butter,  192. 

Yam,  autumn  pigments  of,  58. 

Yellow  Day  Lily,  carotinoids  in,  73. 

Yellow  carotin,'  properties  of  from 
Crustacea,  164. 

Yellow  corn,  effect  on  color  of  butter 
of  feeding  to  cows,  191,  271;  probable 
effect  of  feeding  to  cows  on  vitamin- 
content  of  butter,  271. 

Yellow  xanthophyll  of  Sorby,  iden- 
tity of  with  Tswett's  xantiaophyll 
|3,  44. 

Yew,  autumn  pigments  of,  59;  aril,  pig- 
ments of,  86;  leaves,  carotin  content 
of,  249. 

Y.  Xanthophyll,  41,  42,  70;  blue  color 
reaction  of  with  acids,  42. 

Zea  mays,  87. 

Zoonerythrine,  as  red  pigment  in  bird 
feathers,  143;  in  Crustacea,  162,  165; 
in  echinoderms,  166;  in  fish  skins, 
146;  in  fish  livers,  148;  in  molluscs, 
167;  in  salmon  muscle,  148;  relation 
of  to  rhodophane,  143,  148. 

Zoorubin,  as  red  non-carotinoid  in  bird 
feathers,  143. 


Date  Due 

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